Serpentine transmembrane antigens expressed in human cancers and uses thereof

ABSTRACT

Described is a novel family of cell surface serpentine transmembrane antigens. Two of the proteins in this family are exclusively or predominantly expressed in the prostate, as well as in prostate cancer, and thus members of this family have been termed “STEAP” ( S ix  T ransmembrane  E pithelial  A ntigen of the  P rostate). Four particular human STEAP&#39;s are described and characterized herein. The human STEAP&#39;s exhibit a high degree of structural conservation among them but show no significant structural homology to any known human proteins. The prototype member of the STEAP family, STEAP-1, appears to be a type IIIa membrane protein expressed predominantly in prostate cells in normal human tissues. Structurally, STEAP-1 is a 339 amino acid protein characterized by a molecular topology of six transmembrane domains and intracellular N- and C-termini, suggesting that it folds in a “serpentine” manner into three extracellular and two intracellular loops. STEAP-1 protein expression is maintained at high levels across various stages of prostate cancer. Moreover, STEAP-1 is highly over-expressed in certain other human cancers.

RELATED APPLICATIONS

The application is a continuation of U.S. application Ser. No.09/455,486, filed Dec. 6, 1999, now U.S. Pat. No. 6,833,438 which is acontinuation-in-part of U.S. application Ser. No. 09/323,873, filed Jun.1, 1999, now U.S. Pat. No. 6,329,503, which claims the benefit ofpriority to U.S. provisional application No. 60/087,520, filed Jun. 1,1998, now abandoned, and U.S. provisional application No. 60/091,183,filed Jun. 30, 1998, now abandoned, all of which are hereby incorporatedby reference in their entirety.

TECHNICAL FIELD

The invention described herein relates to a family of novel genes andtheir encoded proteins and tumor antigens, termed STEAP's, and todiagnostic and therapeutic methods and compositions useful in themanagement of various cancers, particularly including prostate cancer,colon cancer, bladder cancer, ovarian cancer and pancreatic cancer.

BACKGROUND ART

Cancer is the second leading cause of human death next to coronarydisease. Worldwide, millions of people die from cancer every year. Inthe United States alone, cancer causes the death of well over ahalf-million people annually, with some 1.4 million new cases diagnosedper year. While deaths from heart disease have been decliningsignificantly, those resulting from cancer generally are on the rise. Inthe early part of the next century, cancer is predicted to become theleading cause of death.

Worldwide, several cancers stand out as the leading killers. Inparticular, carcinomas of the lung, prostate, breast, colon, pancreas,and ovary represent the primary causes of cancer death. These andvirtually all other carcinomas share a common lethal feature. With veryfew exceptions, metastatic disease from a carcinoma is fatal. Moreover,even for those cancer patients who initially survive their primarycancers, common experience has shown that their lives are dramaticallyaltered. Many cancer patients experience strong anxieties driven by theawareness of the potential for recurrence or treatment failure. Manycancer patients experience physical debilitations following treatment,and many experience a recurrence.

Generally speaking, the fundamental problem in the management of thedeadliest cancers is the lack of effective and non-toxic systemictherapies. While molecular medicine promises to redefine the ways inwhich these cancers are managed, progress in this area has been slowdespite intensive worldwide efforts to develop novel moleculardiagnostics and therapeutics. Fundamental to these efforts is the searchfor truly tumor-specific genes and proteins that could be used asdiagnostic and prognostic markers and/or therapeutic targets or agents.

As discussed below, the management of prostate cancer serves as a goodexample of the limited extent to which molecular biology has translatedinto real progress in the clinic. With limited exceptions, the situationis similar for the other major carcinomas mentioned above.

Worldwide, prostate cancer is the fourth most prevalent cancer in men.In North America and Northern Europe, it is by far the most common malecancer and is the second leading cause of cancer death in men. In theUnited States alone, well over 40,000 men die annually of this diseasesecond only to lung cancer. Despite the magnitude of these figures,there is still no effective treatment for metastatic prostate cancer.Surgical prostatectomy, radiation therapy, hormone ablation therapy, andchemotherapy continue to be the main treatment modalities.Unfortunately, these treatments are ineffective for many and are oftenassociated with undesirable consequences.

Most prostate cancers initially occur in the peripheral zone of theprostate gland, away from the urethra. Tumors within this zone may notproduce any symptoms and, as a result, most men with early-stageprostate cancer will not present clinical symptoms of the disease untilsignificant progression has occurred. Tumor progression into thetransition zone of the prostate may lead to urethral obstruction, thusproducing the first symptoms of the disease. However, these clinicalsymptoms are indistinguishable from the common non-malignant conditionof benign prostatic hyperplasia (BPH).

Early detection and diagnosis of prostate cancer currently relies ondigital rectal examinations (DRE), prostate specific antigen (PSA)measurements, transrectal ultrasonography (TRUS), and transrectal needlebiopsy (TRNB). At present, serum PSA measurement in combination with DRErepresent the leading tool used to detect and diagnose prostate cancer.Both have major limitations which have fueled intensive research intofinding better diagnostic markers of this disease.

Accordingly, the lack of a prostate tumor marker that can accuratelydetect early-stage, localized tumors remains a significant limitation inthe management of prostate cancer. A similar problem is the lack of aneffective prognostic marker for determining which cancers are indolentand which ones are or will be aggressive. PSA, for example, cannotaccurately discriminate between these alternatives.

Although the serum PSA assay has been a very useful tool, itsspecificity and general utility is widely regarded as lacking in severalimportant respects. For example, PSA is not a disease-specific marker,as elevated levels of PSA are detectable in a large percentage ofpatients with BPH and prostatitis (25-86%)(Gao, X., et al., Prostate(1997) 31:264-281), as well as in other nonmalignant disorders and insome normal men. Elevations in serum PSA of between 4 to 10 ng/ml areobserved in BPH, and even higher values are observed in prostatitis,particularly acute prostatitis. BPH is an extremely common condition inmen. Further confusing the situation is the fact that serum PSAelevations may be observed without any indication of disease from DRE,and vice versa. In addition, PSA diagnostics have sensitivities of onlybetween 57-79% (Cupp, M. R., et al., Mayo Clin Proc (1993) 68:297-306),and thus miss identifying prostate cancer in a significant population ofmen with the disease. Moreover, it is now recognized that PSA is notprostate-specific (Gao, et al., supra, for review). Various methodsdesigned to improve the specificity of PSA-based detection have beendescribed, such as measuring PSA density and the ratio of free vs.complexed PSA. However, none of these methodologies have been able toreproducibly distinguish benign from malignant prostate disease.

Similarly, there is no available marker that can predict the emergenceof the typically fatal metastatic stage of prostate cancer. Diagnosis ofthe metastatic stage is presently achieved by open surgical orlaparoscopic pelvic lymphadenectomy, whole body radionuclide scans,skeletal radiography, and/or bone lesion biopsy analysis. Clearly,better imaging and less invasive diagnostic methods would improvediagnostic accuracy, ease the burden such procedures place on patients,and open therapeutic options.

There are some known markers which are expressed predominantly inprostate, such as prostate specific membrane antigen (PSM), a hydrolasewith 85% identity to a rat neuropeptidase (Carter, et al., Proc. Natl.Acad. Sci. USA (1996)93:749; Bzdega, et al., J. Neurochem. (1997)69:2270). However, the expression of PSM in small intestine and brain(Israeli, et al., Cancer Res. (1994) 54:1807), as well its potentialrole in neuropeptide catabolism in brain, raises concern of potentialneurotoxicity with anti-PSM therapies. Preliminary results using anIndium-111 labeled, anti-PSM monoclonal antibody to image recurrentprostate cancer show some promise (Sodee, et al., Clin Nuc Med (1996)21:759-766). More recently identified prostate cancer markers includePCTA-1 (Su, et al., Proc. Natl. Acad. Sci. USA (1996) 93:7252) andprostate stem cell antigen (PSCA) (Reiter, et al., Proc. Natl. Acad.Sci. USA (1998) 95:1735). PCTA-1, a novel galectin, is largely secretedinto the media of expressing cells and may hold promise as a diagnosticserum marker for prostate cancer (Su, et al., 1996). PSCA, a GPI-linkedcell surface molecule, was cloned from LAPC-4 cDNA and is unique in thatit is expressed primarily in basal cells of normal prostate tissue andin cancer epithelia (Reiter, et al., 1998). Vaccines for prostate cancerare also being actively explored with a variety of antigens, includingPSM and PSA.

DISCLOSURE OF THE INVENTION

The present invention relates to a novel family of cell surfaceserpentine transmembrane antigens. Two of the proteins in this familyare exclusively or predominantly expressed in the prostate, as well asin prostate cancer, and thus members of this family have been termed“STEAP” (Six Transmembrane Epithelial Antigen of the Prostate). Fourparticular human STEAP's are described and characterized herein. Thehuman STEAP's exhibit a high degree of structural conservation amongthem but show no significant structural homology to any known humanproteins.

The prototype member of the STEAP family, STEAP-1, appears to be a typeIIIa membrane protein expressed predominantly in prostate cells innormal human tissues. Structurally, STEAP-1 is a 339 amino acid proteincharacterized by a molecular topology of six transmembrane domains andintracellular N- and C-termini, suggesting that it folds in a“serpentine” manner into three extracellular and two intracellularloops. STEAP-1 protein expression is maintained at high levels acrossvarious stages of prostate cancer. Moreover, STEAP-1 is highlyover-expressed in certain other human cancers. In particular, cellsurface expression of STEAP-1 has been definitively confirmed in avariety of prostate and prostate cancer cells, bladder cancer cells andcolon cancer cells. These characteristics indicate that STEAP-1 is aspecific cell-surface tumor antigen expressed at high levels inprostate, bladder, colon, and other cancers.

A second member of the family, STEAP-2, is a 454 amino acid protein witha predicted molecular topology similar to that of STEAP-1. STEAP-2, likeSTEAP-1, is prostate-specific in normal human tissues and is alsoexpressed in prostate cancer. Alignment of the STEAP-2 and STEAP-1 ORF'sshows 54.9% identity over a 237 amino acid residue overlap, and thelocations of the six putative transmembrane domains in STEAP-2 coincidewith the locations of the transmembrane domains in STEAP-1 (FIG. 11A).

STEAP-3 and STEAP-4 are also described herein. These are alsostructurally related, and show unique expression profiles. Inparticular, STEAP-3 and STEAP-4 appear to show a different tissuerestriction patterns. An amino acid sequence alignment of all fourSTEAP's is shown in FIG. 11A.

The invention provides polynucleotides corresponding or complementary toall or part of the STEAP genes, mRNA's, and/or coding sequences,preferably in isolated form, including polynucleotides encoding STEAPproteins and fragments thereof, DNA, RNA, DNA/RNA hybrid, and relatedmolecules, polynucleotides or oligonucleotides complementary to theSTEAP genes or mRNA sequences or parts thereof, and polynucleotides oroligonucleotides which hybridize to the STEAP genes, mRNA's, or toSTEAP-encoding polynucleotides. Also provided are means for isolatingcDNA's and the genes encoding STEAP's. Recombinant DNA moleculescontaining STEAP polynucleotides, cells transformed or transduced withsuch molecules, and host-vector systems for the expression of STEAP geneproducts are also provided. The invention further provides STEAPproteins and polypeptide fragments thereof. The invention furtherprovides antibodies that bind to STEAP proteins and polypeptidefragments thereof, including polyclonal and monoclonal antibodies,murine and other mammalian antibodies, chimeric antibodies, humanizedand fully human antibodies, and antibodies labeled with a detectablemarker, and antibodies conjugated to radionuclides, toxins or othertherapeutic compositions. The invention further provides methods fordetecting the presence of STEAP polynucleotides and proteins in variousbiological samples, as well as methods for identifying cells thatexpress a STEAP. The invention further provides various therapeuticcompositions and strategies for treating prostate cancer, includingparticularly, antibody, vaccine and small molecule therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. STEAP-1 structure. 1A-1-1A-2: Nucleotide and deduced amino acidsequences of STEAP-1 (8P1B4) clone 10 cDNA (SEQ ID NOS. 1 and 2,respectively). The start Methionine is indicated in bold at amino acidresidue position 1 and six putative transmembrane domains are indicatedin bold and are underlined. 1B: Schematic representation of STEAP-1transmembrane orientation; amino acid residues bordering the predictedextracellular domains are indicated and correspond to the numberingscheme of FIG. 1A. 1C: G/C rich 5′ non-coding sequence of the STEAP-1gene as determined by overlapping sequences of clone 10 and clone 3 (SEQID NO: 3).

FIG. 2. Predominant expression of STEAP-1 in prostate tissue. Firststrand cDNA was prepared from 16 normal tissues, the LAPC xenografts(4AD, 4AI and 9AD) and HeLa cells. Normalization was performed by PCRusing primers to actin and GAPDH. Semi-quantitative PCR, using primersderived from STEAP-1 (8P1D4) cDNA (FIG. 1A), shows predominantexpression of STEAP-1 in normal prostate and the LAPC xenografts. Thefollowing primers were used to amplify STEAP-1:

8P1D4.1 5′ ACTTTGTTGATGACCAGGATTGGA 3′ (SEQ ID NO: 14) 8P1D4.2 5′CAGAACTTCAGCACACACAGGAAC 3′ (SEQ ID NO: 15)

FIG. 3. Northern blot analyses of STEAP-1 expression in various normalhuman tissues and prostate cancer xenografts, showing predominantexpression of STEAP-1 in prostate tissue. FIG. 3A: Two multiple tissuenorthern blots (Clontech) were probed with a full length STEAP cDNAclone 10 (FIG. 1A; SEQ ID NO: 1). Size standards in kilobases (kb) areindicated on the side. Each lane contains 2 μg of mRNA that wasnormalized by using a β-actin probe. FIG. 3B: Multiple tissue RNA dotblot (Clontech, Human Master Blot cat# 7770-1) probed with STEAP-1 cDNAclone 10 (FIG. 1A; SEQ ID NO: 1), showing approximately five-foldgreater expression in prostate relative to other tissues withsignificant detectable expression.

FIG. 4A-4B. Nucleotide sequence of STEAP-1 GTH9 clone (SEQ ID NO: 4)corresponding to the 4 kb message on northern blots (FIG. 3A). Thesequence contains an intron of 2399 base pairs relative to the STEAP-1clone 10 sequence of FIG. 1A; coding regions are nucleotides 96-857 and3257-3510 (indicated in bold). The start ATG is in bold and underlined,the STOP codon is in bold and underlined, and the intron-exon boundariesare underlined.

FIG. 5. Expression of STEAP-1 in prostate and multiple cancer cell linesand prostate cancer xenografts. Xenograft and cell line filters wereprepared with 10 μg of total RNA per lane. The blots were analyzed usingthe STEAP-1 clone 10 as probe. All RNA samples were normalized byethidium bromide staining and subsequent analysis with a β-actin probe.FIG. 5A: Expression in various cancer cell lines and xenografts andprostate. Lanes as follows: (1) PrEC cells, (2) normal prostate tissue,(3) LAPC-4 AD xenograft, (4) LAPC-4 AI xenograft, (5) LAPC-9 ADxenograft, (6) LAPC-9 AI xenograft, (7) LNCaP cells, (8) PC-3 cells, (9)DU145 cells, (10) PANC-1 cells, (11) B×PC-3 cells, (12) HPAC cells, (13)Capan-1 cells, (14) CACO-2 cells, (15) LOVO cells, (16) T84 cells, (17)COLO-205 cells, (18) KCL-22 cells (acute lymphocytic leukemia, ALL),(19) HT1197 cells, (20) SCABER cells, (21) UM-UC-3 cells, (22) TCCSUPcells, (23) J82 cells, (24) 5637 cells, (25) RD-ES cells (Ewing sarcoma,EWS), (26) CAMA-1 cells, (27) DU4475 cells, (28) MCF-7 cells, (29)MDA-MB-435s cells, (30) NTERA-2 cells, (31) NCCIT cells, (32) TERA-1cells, (33) TERA-2 cells, (34) A431 cells, (35) HeLa cells, (36) OV-1063cells, (37) PA-1 cells, (38) SW 626 cells, (39) CAOV-3 cells. FIG. 5B:The expression of STEAP-1 in subcutaneously (sc) grown LAPC xenograftscompared to the expression in LAPC-4 and LAPC-9 xenografts grown in thetibia (it) of mice.

FIG. 6. Western blot analysis of STEAP-1 protein expression in tissuesand multiple cell lines. Western blots of cell lysates prepared fromprostate cancer xenografts and cell lines were probed with a polyclonalanti-STEAP-1 antibody preparation (see Example 3C for details). Thesamples contain 20 μg of protein and were normalized with anti-Grb-2probing of the Western blots.

FIG. 7. Cell surface biotinylation of STEAP-1. FIG. 7A: Cell surfacebiotinylation of 293T cells transfected with vector alone or with vectorcontaining cDNA encoding 6His-tagged STEAP-1. Cell lysates wereimmunoprecipitated with specific antibodies, transferred to a membraneand probed with horseradish peroxidase-conjugated streptavidin. Lanes1-4 and 6 correspond to immunoprecipitates from lysates prepared fromSTEAP-1 expressing 293T cells. Lanes 5 and 7 are immunoprecipitates fromvector transfected cells. The immunoprecipitations were performed usingthe following antibodies: (1) sheep non-immune, (2) anti-Large Tantigen, (3) anti-CD71 (transferrin receptor), (4) anti-His, (5)anti-His, (6) anti-STEAP-1, (7) anti-STEAP-1. FIG. 7B: Prostate cancer(LNCaP, PC-3, DU145), bladder cancer (UM-UC-3, TCCSUP) and colon cancer(LOVO, COLO) cell lines were either biotinylated (+) or not (−) prior tolysis. Western blots of streptavidin-gel purified proteins were probedwith anti-STEAP-1 antibodies. Molecular weight markers are indicated inkilo Daltons (kD).

FIG. 8. Immunohistochemical analysis of STEAP-1 expression usinganti-STEAP-1 polyclonal antibody. Tissues were fixed in 10% formalin andembedded in paraffin. Tissue sections were stained using anti-STEAP-1polyclonal antibodies directed towards the N-terminal peptide. Samplesinclude: (a) LNCaP cells probed in the presence of N-terminal STEAP-1peptide 1, (b) LNCaP plus non specific peptide 2, (c) normal prostatetissue, (d) grade 3 prostate carcinoma, (e) grade 4 prostate carcinoma,(f) LAPC-9 AD xenograft, (g) normal bladder, (h) normal colon. Allimages are at 400× magnification.

FIG. 9A-9B. Nucleotide and deduced amino acid sequences of STEAP-2(98P4B6) clone GTD3 cDNA (SEQ ID NOS: 5 and 6, respectively). The startmethionine and Kozak sequence are indicated in bold, and the putativetransmembrane domains are underlined in bold. The 5′ UTR exhibits a highGC content of 72%.

FIG. 10A-1-10A-2. Nucleotide and deduced amino acid sequences of STEAP-3(SEQ ID NOS: 7 and 8, respectively). Kozak region is bolded. FIG. 10B.Nucleotide sequences (SEQ ID NOS: 9-12, respectively) of dbEST databaseentries corresponding to additional STEAP family members obtained bysearching with the protein sequence of STEAP-1.

FIG. 11. Primary structural comparisons of STEAP family proteins:

FIG. 11A-1-11B-2. Amino acid sequence alignment of STEAP-1 (8P1D4 clone10; SEQ ID NO:2), STEAP-2 (98P4B6 clone GTD3: SEQ ID NO:6), STEAP-3(98P4B6 clone GTD3; SEQ ID NO:8), and STEAP-4/R80991 (SEQ ID NO:13)using the PIMA 1.4 program; transmembrane domains identified by theSOSUI program are in bold. PIMA maximal linkage clustering resultsshown; identical residues shown in bold.

FIG. 11B. Amino acid sequence alignment of STEAP-1 (8P1D4 clone 10; SEQID NO: 2) and STEAP-2 (98P4B6 clone GTD3; SEQ ID NO: 6) sequences. Thealignment was performed using the SIM alignment program of the BaylorCollege of Medicine Search Launcher Web site. Transmembrane domains areindicated in boldface. The results show a 54.9% identity in a 237residues overlap (Score: 717.0; Gap frequency: 0.0%).

FIG. 11C. Amino acid sequence alignment of STEAP-1 and STEAP-3 (98P4B6clone GTD3; SEQ ID NO: 8) sequences. Identical residues indicated withasterisks. SIM results: 40.9% identity in 264 residues overlap; Score:625.0; Gap frequency: 0.0%.

FIG. 11D. Amino acid sequence alignment of STEAP-2 and STEAP-3 (98P4B6clone GTD3; SEQ ID NO: 8) sequences. Identical residues indicated withasterisks. SIM results: 47.8% identity in 416 residues overlap; Score:1075.0; Gap frequency: 0.2%.

FIG. 12. Expression of STEAP-3 mRNA in normal tissues by Northern blot(FIG. 12A) and RT-PCR (FIG. 12B). For RT-PCR analysis, first strand cDNAwas prepared from 16 normal tissues. Normalization was performed by PCRusing primers to actin and GAPDH. Semi-quantitative PCR, using primersto AI139607, shows predominant expression of AI139607 in placenta andprostate after 25 cycles of amplification. The following primers wereused to amplify AI139607:

(SEQ ID NO: 16) AI139607.1 5′ TTAGGACAACTTGATCACCAGCA 3′ (SEQ ID NO: 17)AI139607.2 5′ TGTCCAGTCCAAACTGGGTTATTT 3′

FIG. 13. Predominant expression of STEAP-4/R80991 in liver. First strandcDNA was prepared from 16 normal tissues. Normalization was performed byPCR using primers to actin and GAPDH. Semi-quantitative PCR, usingprimers to R80991, shows predominant expression of R80991 in liver after25 cycles of amplification. The following primers were used to amplifyR80991:

R80991.1 5′ AGGGAGTTCAGCTTCGTTCAGTC 3′ (SEQ ID NO: 18) R80991.2 5′GGTAGAACTTGTAGCGGCTCTCCT 3′ (SEQ ID NO: 19)

FIG. 14. Predominant expression of STEAP-2 (98P4B6) in prostate tissue.First strand cDNA was prepared from 8 normal tissues, the LAPCxenografts (4AD, 4AI and 9AD) and HeLa cells. Normalization wasperformed by PCR using primers to actin and GAPDH. Semi-quantitativePCR, using primers to 98P4B6, shows predominant expression of 98P4B6 innormal prostate and the LAPC xenografts. The following primers were usedto amplify STEAP II:

98P4B6.1 5′ GACTGAGCTGGAACTGGAATTTGT 3′ (SEQ ID NO: 20) 98P4B6.2 5′TTTGAGGAGACTTCATCTCACTGG 3′ (SEQ ID NO: 21)

FIG. 15. Expression of the prostate-specific STEAP-2/98P4B6 gene innormal tissues and in prostate cancer xenografts determined by Northernblot analysis. Human normal tissue filters (A and B) were obtained fromClontech and contain 2 μg of mRNA per lane. Xenograft filter (C) wasprepared with 10 μg of total RNA per lane. The blots were analyzed usingthe SSH derived 98P4B6 clone as probe. All RNA samples were normalizedby ethidium bromide staining.

FIG. 16. Expression of STEAP-2 in prostate and select cancer cell linesas determined by Northern blot analysis. Xenograft and cell line filterswere prepared with 10 μg total RNA per lane. The blots were analyzedusing an SSH derived 98P4B6 clone as probe. All RNA samples werenormalized by ethidium bromide staining.

FIG. 17. Chromosomal localization of STEAP family members. Thechromosomal localizations of the STEAP genes described herein weredetermined using the GeneBridge4 radiation hybrid panel (ResearchGenetics, Huntsville Ala.). The mapping for STEAP-2 and AI139607 wasperformed using the Stanford G3 radiation hybrid panel (ResearchGenetics, Huntsville Ala.).

FIG. 18. Schematic representation of Intron-Exon boundaries within theORF of human STEAP-1 gene. A total of 3 introns (i) and 4 exons (e) wereidentified.

FIG. 19. Zooblot southern analysis of STEAP-1 gene in various species.Genomic DNA was prepared from several different organisms includinghuman, monkey, dog, mouse, chicken and Drosophila. Ten micrograms ofeach DNA sample was digested with EcoRI, blotted onto nitrocellulose andprobed with a STEAP-1 probe. Size standards are indicated on the side inkilobases (kb).

FIG. 20. Southern blot analysis of mouse BAC with a STEAP-1 probe. DNAwas prepared from human cells to isolate genomic DNA and from a mouseBAC clone (12P11) that contains the mouse STEAP gene. Each DNA samplewas digested with EcoRI, blotted onto nitrocellulose and probed. Eightmicrograms of genomic DNA was compared to 250 ng of mouse BAC DNA.

MODES OF CARRYING OUT THE INVENTION

Unless otherwise defined, all terms of art, notations and otherscientific terminology used herein are intended to have the meaningscommonly understood by those of skill in the art to which this inventionpertains. In some cases, terms with commonly understood meanings aredefined herein for clarity and/or for ready reference, and the inclusionof such definitions herein should not necessarily be construed torepresent a substantial difference over what is generally understood inthe art. The techniques and procedures described or referenced hereinare generally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized molecular cloning methodologies described in Sambrook,et al., Molecular Cloning: A Laboratory Manual, 2nd. edition (1989) ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. As appropriate,procedures involving the use of commercially available kits and reagentsare generally carried out in accordance with manufacturer definedprotocols and/or parameters unless otherwise noted.

As used herein, the terms “advanced prostate cancer”, “locally advancedprostate cancer”, “advanced disease” and “locally advanced disease” meanprostate cancers which have extended through the prostate capsule, andare meant to include stage C disease under the American UrologicalAssociation (AUA) system, stage C1-C2 disease under the Whitmore-Jewettsystem, and stage T3-T4 and N+ disease under the TNM (tumor, node,metastasis) system. In general, surgery is not recommended for patientswith locally advanced disease, and these patients have substantiallyless favorable outcomes compared to patients having clinically localized(organ-confined) prostate cancer. Locally advanced disease is clinicallyidentified by palpable evidence of induration beyond the lateral borderof the prostate, or asymmetry or induration above the prostate base.Locally advanced prostate cancer is presently diagnosed pathologicallyfollowing radical prostatectomy if the tumor invades or penetrates theprostatic capsule, extends into the surgical margin, or invades theseminal vesicles.

As used herein, the terms “metastatic prostate cancer” and “metastaticdisease” mean prostate cancers which have spread to regional lymph nodesor to distant sites, and are meant to include stage D disease under theAUA system and stage T×N×M+ under the TNM system. As is the case withlocally advanced prostate cancer, surgery is generally not indicated forpatients with metastatic disease, and hormonal (androgen ablation)therapy is the preferred treatment modality. Patients with metastaticprostate cancer eventually develop an androgen-refractory state within12 to 18 months of treatment initiation, and approximately half of thesepatients die within 6 months thereafter. The most common site forprostate cancer metastasis is bone. Prostate cancer bone metastases are,on balance, characteristically osteoblastic rather than osteolytic(i.e., resulting in net bone formation). Bone metastases are found mostfrequently in the spine, followed by the femur, pelvis, rib cage, skulland humerus. Other common sites for metastasis include lymph nodes,lung, liver and brain. Metastatic prostate cancer is typically diagnosedby open or laparoscopic pelvic lymphadenectomy, whole body radionuclidescans, skeletal radiography, and/or bone lesion biopsy.

As used herein, the term “polynucleotide” means a polymeric form ofnucleotides of at least 10 bases or base pairs in length, eitherribonucleotides or deoxynucleotides or a modified form of either type ofnucleotide, and is meant to include single and double stranded forms ofDNA.

As used herein, the term “polypeptide” means a polymer of at least 8amino acids. Throughout the specification, standard three letter orsingle letter designations for amino acids are used.

As used herein, the terms “hybridize”, “hybridizing”, “hybridizes” andthe like, used in the context of polynucleotides, are meant to refer toconventional hybridization conditions, preferably such as hybridizationin 50% formamide/6×SSC/0.1% SDS/100 μg/ml ssDNA, in which temperaturesfor hybridization are above 37° C. and temperatures for washing in0.1×SSC/0.1% SDS are above 55° C., and most preferably to stringenthybridization conditions.

In the context of amino acid sequence comparisons, the term “identity”is used to express the percentage of amino acid residues at the samerelative position which are the same. Also in this context, the term“homology” is used to express the percentage of amino acid residues atthe same relative positions which are either identical or are similar,using the conserved amino acid criteria of BLAST analysis, as isgenerally understood in the art. Further details regarding amino acidsubstitutions, which are considered conservative under such criteria,are provided below.

Additional definitions are provided throughout the subsections whichfollow.

Molecular and Biochemical Features of the STEAP's

The invention relates to a novel family of proteins, termed STEAP's.Four STEAP's are specifically described herein by way of structural,molecular and biochemical features. As is further described in theExamples which follow, the STEAP's have been characterized in a varietyof ways. For example, analyses of nucleotide coding and amino acidsequences were conducted in order to identify conserved structuralelements within the STEAP family. Extensive RT-PCR and Northern blotanalyses of STEAP mRNA expression were conducted in order to establishthe range of normal and cancerous tissues expressing the various STEAPmessages. Western blot, immunohistochemical and flow cytometric analysesof STEAP protein expression were conducted to determine proteinexpression profiles, cell surface localization and gross moleculartopology of STEAP.

The prototype member of the STEAP family, STEAP-1, is asix-transmembrane cell surface protein of 339 amino acids with noidentifiable homology to any known human protein. The cDNA nucleotideand deduced amino acid sequences of human STEAP-1 are shown in FIG. 1A.A gross topological schematic of the STEAP-1 protein integrated withinthe cell membrane is shown in FIG. 1B. STEAP-1 expression ispredominantly prostate-specific in normal tissues. Specifically,extensive analysis of STEAP-1 mRNA and protein expression in normalhuman tissues shows that STEAP-1 protein is predominantly expressed inprostate and, to a far smaller degree, in bladder. STEAP-1 mRNA is alsorelatively prostate specific, with only very low level expressiondetected in a few other normal tissues. In cancer, STEAP-1 mRNA andprotein is consistently expressed at high levels in prostate cancer(including androgen-dependent and androgen-independent tumors) andduring all stages of the disease. STEAP-1 is also expressed in othercancers. Specifically, STEAP-1 mRNA is expressed at very high levels inbladder, colon, pancreatic, and ovarian cancer (as well as othercancers). In addition, cell surface expression of STEAP-1 protein hasbeen established in prostate, bladder and colon cancers. Therefore,STEAP-1 has all of the hallmark characteristics of an excellenttherapeutic target for the treatment of certain cancers, includingparticularly prostate, colon and bladder carcinomas.

A second member of the family, STEAP-2, is a 454 amino acid proteinencoded by a distinct gene and having a predicted molecular topologysimilar to that of STEAP-1. The cDNA nucleotide and deduced amino acidsequences of STEAP-2 are shown in FIG. 9. Amino acid alignment of theSTEAP-1 and STEAP-2 sequences show a high degree of structuralconservation (54.9% identity over a 237 amino acid residue overlap, andthe locations of the six putative transmembrane domains in STEAP-1 andSTEAP-2 coincide (FIGS. 11A and 11B). Structural homology between theseSTEAP-1 and STEAP-2 is highest in the regions spanned by the firstputative extracellular loop to the fifth transmembrane domain. However,some significant structural differences between STEAP-1 and STEAP-2 areapparent. For example, STEAP-2 exhibits a 205 a.a. long intracellularN-terminus (compared to 69 a.a. in STEAP-1) and a short 4 a.a.intracellular C-terminus (compared to 26 a.a. in STEAP-1). In addition,both the STEAP-1 and STEAP-2 genes are located on chromosome 7, but ondifferent arms. These differences could imply significant differences infunction and/or interaction with intracellular signaling pathways.

STEAP-2 is expressed only in normal prostate among human tissues tested(FIGS. 14 and 15) and is also expressed in prostate cancer (FIG. 15),and thus shows some similarity in expression profile to STEAP-1.However, STEAP-2 exhibits a different mRNA expression profile relativeto STEAP-1 in prostate cancer samples (compare FIGS. 3 and 15) and inother non-prostate cancers tested (compare FIGS. 5 and 16). Thesedifferences in the expression profiles of STEAP-1 and STEAP-2 suggestthat they are differentially regulated.

STEAP-3 and STEAP-4 appear to be closely related to both STEAP-1 andSTEAP-2 on a structural level, and both appear to be transmembraneproteins as well. STEAP-3 is more related to STEAP-2 (47.8% identity)than to STEAP-1 (40.9% identity). STEAP-3 and STEAP-4 show uniqueexpression profiles. STEAP-3, for example, appears to have an expressionpattern which is predominantly restricted to placenta and, to a smallerdegree, expression is seen in prostate but not in other normal tissuestested. STEAP-4 seems to be expressed predominantly in liver by RT-PCRanalysis. Neither STEAP-3 nor STEAP-4 appear to be expressed in prostatecancer xenografts which exhibit high level STEAP-1 and STEAP-2expression.

Three of the four STEAP's described herein map to human chromosome 7(STEAP-1, -2 and -3). Interestingly, STEAP-1 maps within 7p22 (7p22.3),a large region of allelic gain reported for both primary and recurrentprostate cancers (Visakorpi, T., et al., Cancer Res. (1995) 55:342-347,Nupponen, N. N., et al., American J. Pathol. (1998) 153:141-148),suggesting that up-regulation of STEAP-1 in cancer might include genomicmechanisms. In addition, both STEAP-2 and STEAP-3 locate to chromosome7q21, suggesting that these two genes arose by gene duplication.

The function of the STEAP's are not known. Other cell surface moleculesthat contain six transmembrane domains include ion channels (Dolly, J.O., et al., J. Bioenerg. Biomembr. (1996) 28:231-253) and water channelsor aquaporins (Reizer, J., et al., Crit. Rev. Biochem. Mol. Biol. (1993)28:237-257). Structural studies show that both types of moleculesassemble into tetrameric complexes to form functional channels(Christie, M. J., Clin. Exp. Pharmacol. Physiol. (1995) 22:944-951,Walz, T., et al., Nature (1997) 387:624-627, Cheng, A., et al., Nature(1997) 387:627-630). Immunohistochemical staining of STEAP-1 in theprostate gland seems to be concentrated at the cell-cell boundaries,with less staining detected at the luminal side. This may suggest a rolefor STEAP-1 in tight-junctions, gap-junctions or cell communication andadhesion. In order to test these possibilities, Xenopus oocytes (orother cells) expressing STEAP may be analyzed using voltage-clamp andpatch-clamp experiments to determine if STEAP functions as anion-channel. Oocyte cell volume may also be measured to determine ifSTEAP exhibits water channel properties. If STEAP's function as channelor gap-junction proteins, they may serve as excellent targets forinhibition using, for example, antibodies, small molecules, andpolynucleotides capable of inhibiting expression or function. Therestricted expression pattern in normal tissue, and the high levels ofexpression in cancer tissue suggest that interfering with STEAP functionmay selectively kill cancer cells.

Since the STEAP gene family is predominantly expressed in epithelialtissue, it seems possible that the STEAP proteins function as ionchannels, transport proteins or gap-junction proteins in epithelial cellfunction. Ion channels have been implicated in proliferation andinvasiveness of prostate cancer cells (Lalani, E. N., et al., CancerMetastasis Rev. (1997) 16:29-66). Both rat and human prostate cancercells contain sub-population of cells with higher and lower expressionlevels of sodium channels. Higher levels of sodium channel expressioncorrelate with more aggressive invasiveness in vitro (Smith, P., et al.,FEBS Lett. (1998) 423:19-24). Similarly, it has been shown that aspecific blockade of sodium channels inhibits the invasiveness of PC-3cells in vitro (Laniado, M. E., et al., Am. J. Pathol. (1997)150:1213-1221), while specific inhibition of potassium channels in LNCaPcells inhibited cell proliferation (Skryma, R. N., et al., Prostate(1997) 33:112-122). These reports suggest a role for ion channels inprostate cancer and also demonstrate that small molecules that inhibition channel function may interfere with prostate cancer proliferation.

STEAP Polynucleotides

One aspect of the invention provides polynucleotides corresponding orcomplementary to all or part of a STEAP gene, mRNA, and/or codingsequence, preferably in isolated form, including polynucleotidesencoding a STEAP protein and fragments thereof, DNA, RNA, DNA/RNAhybrid, and related molecules, polynucleotides or oligonucleotidescomplementary to a STEAP gene or mRNA sequence or a part thereof, andpolynucleotides or oligonucleotides which hybridize to a STEAP gene,mRNA, or to a STEAP-encoding polynucleotide (collectively, “STEAPpolynucleotides”). As used herein, STEAP genes and proteins are meant toinclude the STEAP-1, STEAP-2 and STEAP-3 genes and proteins, and thegene and protein corresponding to GenBank Accession number R80991(STEAP-4), and the genes and proteins corresponding to other STEAPproteins and structurally similar variants of the foregoing. Such otherSTEAP proteins and variants will generally have coding sequences whichare highly homologous to the STEAP coding sequences provided herein, andpreferably will share at least about 50% amino acid identity and atleast about 60% amino acid homology (using BLAST criteria), morepreferably sharing 70% or greater homology (using BLAST criteria).

The STEAP family member gene sequences described herein encode STEAPproteins sharing unique highly conserved amino acid sequence domainswhich distinguish them from other proteins. Proteins which include oneor more of these unique highly conserved domains may be related to theSTEAP family members or may represent new STEAP proteins. Referring toFIG. 11A, which is an amino acid sequence alignment of the full STEAP-1,STEAP-2, and STEAP-3 protein sequences as well as the partial STEAP-4sequence, it is clear that the STEAP's are closely related at thestructural level. Referring to FIG. 11B, which is an amino acid sequencealignment of the full STEAP-1 and STEAP-2 protein sequences, closestructural conservation is apparent, particularly in the predictedtransmembrane domains. The STEAP-1 and STEAP-2 sequences share 54.9%identity over a 237 amino acid overlap. Additional amino acid sequencealignments between the STEAP's are shown in FIGS. 11C and 11D. Thesealignments show that STEAP-1 and STEAP-3 are 40.9% identical over a 264amino acid region, while STEAP-2 and STEAP-3 are 47.8% identical over a416 amino acid region.

A STEAP polynucleotide may comprise a polynucleotide having thenucleotide sequence of human STEAP-1 as shown in FIG. 1A, the nucleotidesequence of human STEAP-2 as shown in FIG. 9, the nucleotide sequence ofhuman STEAP-3 as shown in FIG. 10A, or the nucleotide sequence ofSTEAP-4 as shown in FIG. 10B, or a sequence complementary thereto, or apolynucleotide fragment of any of the foregoing. Another embodimentcomprises a polynucleotide which encodes the human STEAP-1, STEAP-2,STEAP-3 or STEAP-4 protein amino acid sequences, a sequencecomplementary thereto, or a polynucleotide fragment of any of theforegoing. Another embodiment comprises a polynucleotide which iscapable of hybridizing under stringent hybridization conditions to thehuman STEAP-1 cDNA shown in FIG. 1A, the human STEAP-2 cDNA shown inFIG. 9, the human STEAP-3 cDNA shown in FIG. 10A, or the STEAP-4 asshown in FIG. 10B, or to a polynucleotide fragment thereof.

Specifically contemplated are genomic DNA, cDNA's, ribozymes, andantisense molecules, as well as nucleic acid molecules based on analternative backbone or including alternative bases, whether derivedfrom natural sources or synthesized. For example, antisense moleculescan be RNA's or other molecules, including peptide nucleic acids (PNA's)or non-nucleic acid molecules such as phosphorothioate derivatives, thatspecifically bind DNA or RNA in a base pair-dependent manner. Theskilled person can readily obtain these classes of nucleic acidmolecules using the STEAP polynucleotides and polynucleotide sequencesdisclosed herein.

Further specific embodiments of this aspect of the invention includeprimers and primer pairs, which allow the specific amplification of thepolynucleotides of the invention or of any specific parts thereof, andprobes that selectively or specifically hybridize to nucleic acidmolecules of the invention or to any part thereof. Probes may be labeledwith a detectable marker, such as, for example, a radioisotope,fluorescent compound, bioluminescent compound, a chemiluminescentcompound, metal chelator or enzyme. Such probes and primers can be usedto detect the presence of a STEAP polynucleotide in a sample and as ameans for detecting a cell expressing a STEAP protein. Examples of suchprobes include polynucleotides comprising all or part of the humanSTEAP-1, STEAP-2 and STEAP-3 cDNA sequences shown in FIGS. 1A, 9 and10A, respectively. Examples of primer pairs capable of specificallyamplifying STEAP mRNA's are also described in the Examples which follow.As will be understood by the skilled person, a great many differentprimers and probes may be prepared based on the sequences provided inherein and used effectively to amplify and/or detect a STEAP mRNA or anmRNA encoding a particular STEAP family member.

As used herein, a polynucleotide is said to be “isolated” when it issubstantially separated from contaminant polynucleotides whichcorrespond or are complementary to genes other than the STEAP gene fromwhich the polynucleotide is derived or which encode polypeptides otherthan the corresponding STEAP gene product or fragment thereof. Theskilled person can readily employ nucleic acid isolation procedures toobtain an isolated STEAP polynucleotides.

The STEAP polynucleotides of the invention are useful for a variety ofpurposes, including but not limited to their use as probes and primersfor the amplification and/or detection of the STEAP gene(s), mRNA(s), orfragments thereof; as reagents for the diagnosis and/or prognosis ofprostate cancer and other cancers; as coding sequences capable ofdirecting the expression of STEAP polypeptides; as tools for modulatingor inhibiting the expression of the STEAP gene(s) and/or translation ofthe STEAP transcript(s); and as therapeutic agents.

Methods for Isolating STEAP-Encoding Nucleic Acid Molecules

The STEAP cDNA sequences described herein enable the isolation of otherpolynucleotides encoding STEAP gene product(s), as well as the isolationof polynucleotides encoding STEAP gene product homologues, alternativelyspliced isoforms, allelic variants, and mutant forms of the STEAP geneproduct. Various molecular cloning methods that can be employed toisolate full length cDNA's encoding a STEAP gene are well known (See,for example, Sambrook, J., et al., Molecular Cloning: A LaboratoryManual, 2d edition., Cold Spring Harbor Press, New York, 1989; CurrentProtocols in Molecular Biology, Ausubel, et al., Eds., Wiley and Sons,1995). For example, lambda phage cloning methodologies may beconveniently employed, using commercially available cloning systems(e.g., Lambda ZAP Express, Stratagene). Phage clones containing STEAPgene cDNA's may be identified by probing with a labeled STEAP cDNA or afragment thereof. For example, in one embodiment, the STEAP-1 cDNA (FIG.1A) or a portion thereof can be synthesized and used as a probe toretrieve overlapping and full length cDNA's corresponding to a STEAPgene. Similarly, the STEAP-2 and STEAP-3 cDNA sequences may be employed.A STEAP gene may be isolated by screening genomic DNA libraries,bacterial artificial chromosome libraries (BAC's), yeast artificialchromosome libraries (YAC's), and the like, with STEAP DNA probes orprimers.

Recombinant DNA Molecules and Host-Vector Systems

The invention also provides recombinant DNA or RNA molecules containinga STEAP polynucleotide, including but not limited to phages, plasmids,phagemids, cosmids, YAC's, BAC's, as well as various viral and non-viralvectors well known in the art, and cells transformed or transfected withsuch recombinant DNA or RNA molecules. As used herein, a recombinant DNAor RNA molecule is a DNA or RNA molecule that has been subjected tomolecular manipulation in vitro. Methods for generating such moleculesare well known (see, for example, Sambrook, et al. (1989) supra).

The invention further provides a host-vector system comprising arecombinant DNA molecule containing a STEAP polynucleotide within asuitable prokaryotic or eukaryotic host cell. Examples of suitableeukaryotic host cells include a yeast cell, a plant cell, or an animalcell, such as a mammalian cell or an insect cell (e.g., abaculovirus-infectible cell such as an Sf9 cell). Examples of suitablemammalian cells include various prostate cancer cell lines such LnCaP,PC-3, DU145, LAPC-4, TsuPr1, other transfectable or transducibleprostate cancer cell lines, as well as a number of mammalian cellsroutinely used for the expression of recombinant proteins (e.g., COS,CHO, 293, 293T cells). More particularly, a polynucleotide comprisingthe coding sequence of a STEAP may be used to generate STEAP proteins orfragments thereof using any number of host-vector systems routinely usedand widely known in the art.

A wide range of host-vector systems suitable for the expression of STEAPproteins or fragments thereof are available, see, for example, Sambrook,et al. (1989) supra; Current Protocols in Molecular Biology (1995)supra). Preferred vectors for mammalian expression include but are notlimited to pcDNA 3.1 myC-His-tag (Invitrogen) and the retroviral vectorpSRαtkneo (Muller, A. J., et al., Mol. Cell. Biol. (1991) 11:1785).Using these expression vectors, STEAP may be preferably expressed inseveral prostate cancer and non-prostate cell lines, including forexample 293, 293T, rat-1, 3T3, PC-3, LNCaP and TsuPr1. The host-vectorsystems of the invention are useful for the production of a STEAPprotein or fragment thereof. Such host-vector systems may be employed tostudy the functional properties of STEAP and STEAP mutations.

Proteins encoded by the STEAP genes, or by fragments thereof, will havea variety of uses, including but not limited to generating antibodiesand in methods for identifying ligands and other agents and cellularconstituents that bind to a STEAP gene product. Antibodies raisedagainst a STEAP protein or fragment thereof may be useful in diagnosticand prognostic assays, imaging methodologies (including, particularly,cancer imaging), and therapeutic methods in the management of humancancers characterized by expression of a STEAP protein, such asprostate, colon, breast, cervical and bladder carcinomas, ovariancancers, testicular cancers and pancreatic cancers. Variousimmunological assays useful for the detection of STEAP proteins arecontemplated, including but not limited to various types ofradioimmunoassays, enzyme-linked immunosorbent assays (ELISA),enzyme-linked immunofluorescent assays (ELIFA), immunocytochemicalmethods, and the like. Such antibodies may be labeled and used asimmunological imaging reagents capable of detecting prostate cells(e.g., in radioscintigraphic imaging methods). STEAP proteins may alsobe particularly useful in generating cancer vaccines, as furtherdescribed below.

STEAP Proteins

Another aspect of the present invention provides various STEAP proteinsand polypeptide fragments thereof. As used herein, a STEAP proteinrefers to a protein that has or includes the amino acid sequence ofhuman STEAP-1 as provided in FIG. 1A, human STEAP-2 as provided in FIG.9, human STEAP-3 as provided in FIG. 10A, the amino acid sequence ofother mammalian STEAP homologues (e.g., STEAP-4) and variants, as wellas allelic variants and conservative substitution mutants of theseproteins that have STEAP biological activity.

The STEAP proteins of the invention include those specificallyidentified herein, as well as allelic variants, conservativesubstitution variants and homologs that can be isolated/generated andcharacterized without undue experimentation following the methodsoutlined below. Fusion proteins which combine parts of different STEAPproteins or fragments thereof, as well as fusion proteins of a STEAPprotein and a heterologous polypeptide are also included. Such STEAPproteins will be collectively referred to as the STEAP proteins, theproteins of the invention, or STEAP. As used herein, the term “STEAPpolypeptide” refers to a polypeptide fragment or a STEAP protein of atleast 8 amino acids, preferably at least 10 amino acids.

A specific embodiment of a STEAP protein comprises a polypeptide havingthe amino acid sequence of human STEAP-1 as shown in FIG. 1A. Anotherembodiment of a STEAP protein comprises a polypeptide containing theSTEAP-2 amino acid sequence as shown in FIG. 9. Another embodimentcomprises a polypeptide containing the STEAP-3 amino acid sequence ofshown in FIG. 10A. Yet another embodiment comprises a polypeptidecontaining the partial STEAP-4 amino acid sequence of shown in FIG. 11A.

In general, naturally occurring allelic variants of individual humanSTEAP's will share a high degree of structural identity and homology(e.g., 90% or more identity). Typically, allelic variants of the STEAPproteins will contain conservative amino acid substitutions within theSTEAP sequences described herein or will contain a substitution of anamino acid from a corresponding position in a STEAP homologue. One classof STEAP allelic variants will be proteins that share a high degree ofhomology with at least a small region of a particular STEAP amino acidsequence, but will further contain a radical departure form thesequence, such as a non-conservative substitution, truncation, insertionor frame shift. Such alleles may represent mutant STEAP proteins thattypically do not perform the same biological functions or do not haveall of the biological characteristics.

Conservative amino acid substitutions can frequently be made in aprotein without altering either the conformation or the function of theprotein. Such changes include substituting any of isoleucine (I), valine(V), and leucine (L) for any other of these hydrophobic amino acids;aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q)for asparagine (N) and vice versa; and serine (S) for threonine (T) andvice versa. Other substitutions can also be considered conservative,depending on the environment of the particular amino acid and its rolein the three-dimensional structure of the protein. For example, glycine(G) and alanine (A) can frequently be interchangeable, as can alanine(A) and valine (V). Methionine (M), which is relatively hydrophobic, canfrequently be interchanged with leucine and isoleucine, and sometimeswith valine. Lysine (K) and arginine (R) are frequently interchangeablein locations in which the significant feature of the amino acid residueis its charge and the differing pK's of these two amino acid residuesare not significant. Still other changes can be considered“conservative” in particular environments.

STEAP proteins may be embodied in many forms, preferably in isolatedform. As used herein, a protein is said to be “isolated” when physical,mechanical or chemical methods are employed to remove the STEAP proteinfrom cellular constituents that are normally associated with theprotein. A skilled artisan can readily employ standard purificationmethods to obtain an isolated STEAP protein. A purified STEAP proteinmolecule will be substantially free of other proteins or molecules whichimpair the binding of STEAP to antibody or other ligand. The nature anddegree of isolation and purification will depend on the intended use.Embodiments of a STEAP protein include a purified STEAP protein and afunctional, soluble STEAP protein. In one form, such functional, solubleSTEAP proteins or fragments thereof retain the ability to bind antibodyor other ligand.

The invention also provides STEAP polypeptides comprising biologicallyactive fragments of the STEAP amino acid sequence, such as a polypeptidecorresponding to part of the amino acid sequences for STEAP-1 as shownin FIG. 1A, STEAP-2 as shown in FIG. 9, STEAP-3 as shown in FIG. 10A, orSTEAP-4 as shown in FIG. 11A. Such polypeptides of the invention exhibitproperties of a STEAP protein, such as the ability to elicit thegeneration of antibodies which specifically bind an epitope associatedwith a STEAP protein. Polypeptides comprising amino acid sequences whichare unique to a particular STEAP protein (relative to other STEAPproteins) may be used to generate antibodies which will specificallyreact with that particular STEAP protein. For example, referring to theamino acid alignment of the STEAP structures shown in FIGS. 11A-11D, theskilled artisan will readily appreciate that each molecule containsstretches of sequence unique to its structure. These unique stretchescan be used to generate antibodies specific to a particular STEAP.Similarly, regions of conserved sequence may be used to generateantibodies that may bind to multiple STEAP's.

STEAP polypeptides can be generated using standard peptide synthesistechnology or using chemical cleavage methods well known in the artbased on the amino acid sequences of the human STEAP proteins disclosedherein. Alternatively, recombinant methods can be used to generatenucleic acid molecules that encode a polypeptide fragment of a STEAPprotein. In this regard, the STEAP-encoding nucleic acid moleculesdescribed herein provide means for generating defined polypeptidefragments of STEAP proteins. Such STEAP polypeptides or peptides areparticularly useful for generating and characterizing domain specificantibodies (e.g., antibodies recognizing an extracellular orintracellular epitope of a STEAP protein), generating STEAP familymember specific antibodies (e.g., anti-STEAP-1, anti-STEAP-2antibodies), identifying agents or cellular factors that bind to aparticular STEAP or STEAP domain, and in various therapeutic contexts,including but not limited to cancer vaccines. STEAP polypeptidescontaining particularly interesting structures can be predicted and/oridentified using various analytical techniques well known in the art,including, for example, the methods of Chou-Fasman, Garnier-Robson,Kyte-Doolittle, Eisenberg, Karplus-Schultz or Jameson-Wolf analysis, oron the basis of immunogenicity. Fragments containing such structures areparticularly useful in generating subunit specific anti-STEAP antibodiesor in identifying cellular factors that bind to STEAP.

STEAP Antibodies.

Another aspect of the invention provides antibodies that bind to STEAPproteins and polypeptides. The most preferred antibodies willspecifically bind to a STEAP protein and will not bind (or will bindweakly) to non-STEAP proteins and polypeptides. Anti-STEAP antibodiesthat are particularly contemplated include monoclonal and polyclonalantibodies as well as fragments containing the antigen binding domainand/or one or more complementarity determining regions of theseantibodies. As used herein, an antibody fragment is defined as at leasta portion of the variable region of the immunoglobulin molecule whichbinds to its target, i.e., the antigen binding region.

For some applications, it may be desirable to generate antibodies whichspecifically react with a particular STEAP protein and/or an epitopewithin a particular structural domain. For example, preferred antibodiesuseful for cancer therapy and diagnostic imaging purposes are thosewhich react with an epitope in an extracellular region of the STEAPprotein as expressed in cancer cells. Such antibodies may be generatedby using the STEAP proteins described herein, or using peptides derivedfrom predicted extracellular domains thereof, as an immunogen. In thisregard, with reference to the STEAP-1 protein topological schematicshown in FIG. 1B, regions in the extracellular loops between theindicated transmembrane domains may be selected as used to designappropriate immunogens for raising extracellular-specific antibodies.

STEAP antibodies of the invention may be particularly useful in prostatecancer therapeutic strategies, diagnostic and prognostic assays, andimaging methodologies. The invention provides various immunologicalassays useful for the detection and quantification of STEAP and mutantSTEAP proteins and polypeptides. Such assays generally comprise one ormore STEAP antibodies capable of recognizing and binding a STEAP ormutant STEAP protein, as appropriate, and may be performed withinvarious immunological assay formats well known in the art, including butnot limited to various types of radioimmunoassays, enzyme-linkedimmunosorbent assays (ELISA), enzyme-linked immunofluorescent assays(ELIFA), and the like. In addition, immunological imaging methodscapable of detecting prostate cancer are also provided by the invention,including but limited to radioscintigraphic imaging methods usinglabeled STEAP antibodies. Such assays may be clinically useful in thedetection, monitoring, and prognosis of prostate cancer, particularlyadvanced prostate cancer.

The invention also provides various immunological assays useful for thedetection and quantification of STEAP and mutant STEAP proteins andpolypeptides. Such assays generally comprise one or more STEAPantibodies capable of recognizing and binding a STEAP or mutant STEAPprotein, as appropriate, and may be performed within variousimmunological assay formats well known in the art, including but notlimited to various types of radioimmunoassays, enzyme-linkedimmunosorbent assays (ELISA), enzyme-linked immunofluorescent assays(ELIFA), and the like. In addition, immunological imaging methodscapable of detecting prostate cancer and other cancers expressing STEAP(e.g., breast cancer) are also provided by the invention, including butlimited to radioscintigraphic imaging methods using labeled STEAPantibodies. Such assays may be clinically useful in the detection,monitoring, and prognosis of STEAP expressing cancers such as prostatecancer.

STEAP antibodies may also be used in methods for purifying STEAP andmutant STEAP proteins and polypeptides and for isolating STEAPhomologues and related molecules. For example, in one embodiment, themethod of purifying a STEAP protein comprises incubating a STEAPantibody, which has been coupled to a solid matrix, with a lysate orother solution containing STEAP under conditions which permit the STEAPantibody to bind to STEAP; washing the solid matrix to eliminateimpurities; and eluting the STEAP from the coupled antibody. Other usesof the STEAP antibodies of the invention include generatinganti-idiotypic antibodies that mimic the STEAP protein.

Various methods for the preparation of antibodies are well known in theart. For example, antibodies may be prepared by immunizing a suitablemammalian host using a STEAP protein, peptide, or fragment, in isolatedor immunoconjugated form (Antibodies: A Laboratory Manual, CSH Press,Eds., Harlow, and Lane (1988); Harlow, Antibodies, Cold Spring HarborPress, NY (1989)). In addition, fusion proteins of STEAP may also beused, such as a STEAP GST-fusion protein. In a particular embodiment, aGST fusion protein comprising all or most of the open reading frameamino acid sequence of a STEAP may be produced and used as an immunogento generate appropriate antibodies. In another embodiment, a STEAPpeptide may be synthesized and used as an immunogen. As described inExample 5, below, the 15-mer STEAP peptide HSSKEKLRRERIKYC wasconjugated to keyhole limpet hemocyanin (KLH) and used to immunize arabbit. The resulting polyclonal antiserum specifically recognized STEAPexpressed in a recombinant mammalian expression system.

In addition, naked DNA immunization techniques known in the art may beused (with or without purified STEAP protein or STEAP expressing cells)to generate an immune response to the encoded immunogen (for review, seeDonnelly, et al., Ann. Rev. Immunol. (1997) 15:617-648).

The amino acid sequences of the STEAP's provided herein may be used toselect specific regions of the STEAP protein for generating antibodies.For example, hydrophobicity and hydrophilicity analyses of the STEAPamino acid sequence may be used to identify hydrophilic regions in theSTEAP structure. Regions of the STEAP protein that show immunogenicstructure, as well as other regions and domains, can readily beidentified using various other methods known in the art, such asChou-Fasman, Gamier-Robson, Kyte-Doolittle, Eisenberg, Karplus-Schultzor Jameson-Wolf analysis. Methods for the generation of STEAP antibodiesare further illustrated by way of the examples provided herein.

Methods for preparing a protein or polypeptide for use as an immunogenand for preparing immunogenic conjugates of a protein with a carriersuch as BSA, KLH, or other carrier proteins are well known in the art.In some circumstances, direct conjugation using, for example,carbodiimide reagents may be used; in other instances linking reagentssuch as those supplied by Pierce Chemical Co., Rockford, Ill., may beeffective. Administration of a STEAP immunogen is conducted generally byinjection over a suitable time period and with use of a suitableadjuvant, as is generally understood in the art. During the immunizationschedule, titers of antibodies can be taken to determine adequacy ofantibody formation.

STEAP monoclonal antibodies are preferred and may be produced by variousmeans well known in the art. For example, immortalized cell lines whichsecrete a desired monoclonal antibody may be prepared using the standardhybridoma technology of Kohler and Milstein or modifications whichimmortalize producing B cells, as is generally known. The immortalizedcell lines secreting the desired antibodies are screened by immunoassayin which the antigen is the STEAP protein or a STEAP fragment. When theappropriate immortalized cell culture secreting the desired antibody isidentified, the cells may be expanded and antibodies produced eitherfrom in vitro cultures or from ascites fluid.

The antibodies or fragments may also be produced, using currenttechnology, by recombinant means. Regions that bind specifically to thedesired regions of the STEAP protein can also be produced in the contextof chimeric or CDR grafted antibodies of multiple species origin.Humanized or human STEAP antibodies may also be produced and arepreferred for use in therapeutic contexts. Methods for humanizing murineand other non-human antibodies by substituting one or more of thenon-human antibody CDR's for corresponding human antibody sequences arewell known (see, for example, Jones, et al., Nature (1986) 321:522-525;Reichmann, et al., Nature (1988) 332:323-327; Verhoeyan, et al., Science(1988) 239:1534-1536). See also, Carter, et al., Proc. Natl. Acad. Sci.USA (1993) 89:4285 and Sims, et al., J. Immunol. (1993) 151:2296.Methods for producing fully human monoclonal antibodies include phagedisplay and transgenic methods (for review, see Vaughan, et al., NatureBiotechnology (1998) 16:535-539).

Fully human STEAP monoclonal antibodies may be generated using cloningtechnologies employing large human Ig gene combinatorial libraries(i.e., phage display) (Griffiths and Hoogenboom, “Building an in vitroimmune system: human antibodies from phage display libraries.” In:Protein Engineering of Antibody Molecules for Prophylactic andTherapeutic Applications in Man. Clark, M. (Ed.), Nottingham Academic,pp. 45-64 (1993); Burton and Barbas, “Human Antibodies fromcombinatorial libraries.” Id., pp. 65-82). Fully human STEAP monoclonalantibodies may also be produced using transgenic mice engineered tocontain human immunoglobulin gene loci as described in PCT PatentApplication WO 98/24893, Kucherlapati, R., and Jakobovits, A., et al.,published Dec. 3, 1997 (see also, Jakobovits, A., Exp. Opin. Invest.Drugs (1998) 7:607-614). This method avoids the in vitro manipulationrequired with phage display technology and efficiently produces highaffinity authentic human antibodies.

Reactivity of STEAP antibodies with a STEAP protein may be establishedby a number of well known means, including Western blot,immunoprecipitation, ELISA, and FACS analyses using, as appropriate,STEAP proteins, peptides, STEAP-expressing cells or extracts thereof.

A STEAP antibody or fragment thereof of the invention may be labeledwith a detectable marker or conjugated to a second molecule. Suitabledetectable markers include, but are not limited to, a radioisotope, afluorescent compound, a bioluminescent compound, chemiluminescentcompound, a metal chelator or an enzyme. Further, bi-specific antibodiesspecific for two or more STEAP epitopes may be generated using methodsgenerally known in the art. Homodimeric antibodies may also be generatedby cross-linking techniques known in the art (e.g., Wolff, E. A., etal., Cancer Res. (1993) 53:2560-2565).

Methods for the Detection of STEAP

Another aspect of the present invention relates to methods for detectingSTEAP polynucleotides and STEAP proteins, as well as methods foridentifying a cell which expresses STEAP.

More particularly, the invention provides assays for the detection ofSTEAP polynucleotides in a biological sample, such as serum, bone,prostate, and other tissues, urine, semen, cell preparations, and thelike. Detectable STEAP polynucleotides include, for example, a STEAPgene or fragments thereof, STEAP mRNA, alternative splice variant STEAPmRNA's, and recombinant DNA or RNA molecules containing a STEAPpolynucleotide. A number of methods for amplifying and/or detecting thepresence of STEAP polynucleotides are well known in the art and may beemployed in the practice of this aspect of the invention.

In one embodiment, a method for detecting a STEAP mRNA in a biologicalsample comprises producing cDNA from the sample by reverse transcriptionusing at least one primer; amplifying the cDNA so produced using a STEAPpolynucleotides as sense and antisense primers to amplify STEAP cDNA'stherein; and detecting the presence of the amplified STEAP cDNA. Inanother embodiment, a method of detecting a STEAP gene in a biologicalsample comprises first isolating genomic DNA from the sample; amplifyingthe isolated genomic DNA using STEAP polynucleotides as sense andantisense primers to amplify the STEAP gene therein; and detecting thepresence of the amplified STEAP gene. Any number of appropriate senseand antisense probe combinations may be designed from the nucleotidesequences provided for STEAP-1 (FIG. 1A), STEAP-2 (FIG. 9), STEAP-3(FIG. 10A), or STEAP-4 (FIG. 10B), as appropriate, and used for thispurpose.

The invention also provides assays for detecting the presence of a STEAPprotein in a tissue of other biological sample such as serum, bone,prostate, and other tissues, urine, cell preparations, and the like.Methods for detecting a STEAP protein are also well known and include,for example, immunoprecipitation, immunohistochemical analysis, WesternBlot analysis, molecular binding assays, ELISA, ELIFA and the like.

For example, in one embodiment, a method of detecting the presence of aSTEAP protein in a biological sample comprises first contacting thesample with a STEAP antibody, a STEAP-reactive fragment thereof, or arecombinant protein containing an antigen binding region of a STEAPantibody; and then detecting the binding of STEAP protein in the samplethereto.

Methods for identifying a cell which expresses STEAP are also provided.In one embodiment, an assay for identifying a cell which expresses aSTEAP gene comprises detecting the presence of STEAP mRNA in the cell.Methods for the detection of particular mRNA's in cells are well knownand include, for example, hybridization assays using complementary DNAprobes (such as in situ hybridization using labeled STEAP riboprobes,Northern blot and related techniques) and various nucleic acidamplification assays (such as RT-PCR using complementary primersspecific for STEAP, and other amplification type detection methods, suchas, for example, branched DNA, SISBA, TMA and the like). Alternatively,an assay for identifying a cell which expresses a STEAP gene comprisesdetecting the presence of STEAP protein in the cell or secreted by thecell. Various methods for the detection of proteins are well known inthe art and may be employed for the detection of STEAP proteins andSTEAP expressing cells.

STEAP expression analysis may also be useful as a tool for identifyingand evaluating agents which modulate STEAP gene expression. For example,STEAP-1 expression is significantly upregulated in colon, bladder,pancreatic, ovarian and other cancers. Identification of a molecule orbiological agent that could inhibit STEAP-1 over-expression may be oftherapeutic value in the treatment of cancer. Such an agent may beidentified by using a screen that quantifies STEAP expression by RT-PCR,nucleic acid hybridization or antibody binding.

Assays for Determining STEAP Expression Status

Determining the status of STEAP expression patterns in an individual maybe used to diagnose cancer and may provide prognostic information usefulin defining appropriate therapeutic options. Similarly, the expressionstatus of STEAP may provide information useful for predictingsusceptibility to particular disease stages, progression, and/or tumoraggressiveness. The invention provides methods and assays fordetermining STEAP expression status and diagnosing cancers which expressSTEAP.

In one aspect, the invention provides assays useful in determining thepresence of cancer in an individual, comprising detecting a significantincrease in STEAP mRNA or protein expression in a test cell or tissuesample relative to expression levels in the corresponding normal cell ortissue. In one embodiment, the presence of STEAP-1 mRNA is evaluated intissue samples of the colon, pancreas, bladder, ovary, cervix, testis orbreast. The presence of significant STEAP-1 expression in any of thesetissues may be useful to indicate the emergence, presence and/orseverity of these cancers, since the corresponding normal tissues do notexpress STEAP-1 mRNA. In a related embodiment, STEAP-1 expression statusmay be determined at the protein level rather than at the nucleic acidlevel. For example, such a method or assay would comprise determiningthe level of STEAP-1 protein expressed by cells in a test tissue sampleand comparing the level so determined to the level of STEAP expressed ina corresponding normal sample. In one embodiment, the presence ofSTEAP-1 protein is evaluated, for example, using immunohistochemicalmethods. STEAP antibodies or binding partners capable of detecting STEAPprotein expression may be used in a variety of assay formats well knownin the art for this purpose.

Peripheral blood may be conveniently assayed for the presence of cancercells, including prostate, colon, pancreatic, bladder and ovariancancers, using RT-PCR to detect STEAP-1 expression. The presence ofRT-PCR amplifiable STEAP-1 mRNA provides an indication of the presenceof one of these types of cancer. RT-PCR detection assays for tumor cellsin peripheral blood are currently being evaluated for use in thediagnosis and management of a number of human solid tumors. In theprostate cancer field, these include RT-PCR assays for the detection ofcells expressing PSA and PSM (Verkaik, N. S., et al., Urol. Res. (1997)25:373-384; Ghossein, R. A., et al., J. Clin. Oncol. (1995)13:1195-2000; Heston, W. D., et al., Clin. Chem. (1995) 41:1687-1688).RT-PCR assays are well known in the art.

In another approach, a recently described sensitive assay for detectingand characterizing carcinoma cells in blood may be used (Racila, E., etal., Proc. Natl. Acad. Sci. USA (1998) 95:4589-4594). This assaycombines immunomagnetic enrichment with multiparameter flow cytometricand immunohistochemical analyses, and is highly sensitive for thedetection of cancer cells in blood, reportedly capable of detecting oneepithelial cell in 1 ml of peripheral blood.

A related aspect of the invention is directed to predictingsusceptibility to developing cancer in an individual. In one embodiment,a method for predicting susceptibility to cancer comprises detectingSTEAP mRNA or STEAP protein in a tissue sample, its presence indicatingsusceptibility to cancer, wherein the degree of STEAP mRNA expressionpresent is proportional to the degree of susceptibility.

Yet another related aspect of the invention is directed to methods forgauging tumor aggressiveness. In one embodiment, a method for gaugingaggressiveness of a tumor comprises determining the level of STEAP mRNAor STEAP protein expressed by cells in a sample of the tumor, comparingthe level so determined to the level of STEAP mRNA or STEAP proteinexpressed in a corresponding normal tissue taken from the sameindividual or a normal tissue reference sample, wherein the degree ofSTEAP mRNA or STEAP protein expression in the tumor sample relative tothe normal sample indicates the degree of aggressiveness.

Methods for detecting and quantifying the expression of STEAP mRNA orprotein are described herein and use standard nucleic acid and proteindetection and quantification technologies well known in the art.Standard methods for the detection and quantification of STEAP mRNAinclude in situ hybridization using labeled STEAP riboprobes, Northernblot and related techniques using STEAP polynucleotide probes, RT-PCRanalysis using primers specific for STEAP, and other amplification typedetection methods, such as, for example, branched DNA, SISBA, TMA andthe like. In a specific embodiment, semi-quantitative RT-PCR may be usedto detect and quantify STEAP mRNA expression as described in theExamples which follow. Any number of primers capable of amplifying STEAPmay be used for this purpose, including but not limited to the variousprimer sets specifically described herein. Standard methods for thedetection and quantification of protein may be used for this purpose. Ina specific embodiment, polyclonal or monoclonal antibodies specificallyreactive with the wild-type STEAP protein may be used in animmunohistochemical assay of biopsied tissue.

Diagnostic Imaging of Human Cancers

Antibodies specific for STEAP's may be particularly useful inradionuclide and other forms of diagnostic imaging of certain cancers,given their expression profiles and cell surface location. For example,immunohistochemical analysis of STEAP-1 protein suggests that in normaltissues STEAP-1 is predominantly restricted to prostate and bladder. Thetransmembrane orientation of STEAP-1 (and presumably STEAP-2, STEAP-3,STEAP-4) provides a target readily identifiable by antibodiesspecifically reactive with extracellular epitopes. This tissuerestricted expression, and the localization of STEAP to the cell surfaceof multiple cancers makes STEAP an ideal candidate for diagnosticimaging. Accordingly, in vivo imaging techniques may be used to imagehuman cancers expressing a STEAP protein.

For example, cell surface STEAP-1 protein is expressed at very highlevels in several human cancers, particularly prostate, bladder, colonand ovarian cancers, and Ewing sarcoma. Moreover, in normal tissues,STEAP-1 protein expression is largely restricted to prostate. Thus,radiolabeled antibodies specifically reactive with extracellularepitopes of STEAP-1 may be particularly useful in vivo imaging of solidtumors of the foregoing cancers. Such labeled anti-STEAP-1 antibodiesmay provide very high level sensitivities for the detection ofmetastasis of these cancers.

Preferably, monoclonal antibodies are used in the diagnostic imagingmethods of the invention.

Cancer Immunotherapy and Cancer Vaccines

The invention provides various immunotherapeutic methods for treatingprostate cancer, including antibody therapy, in vivo vaccines, and exvivo immunotherapy methods, which utilize polynucleotides andpolypeptides corresponding to STEAP and STEAP antibodies. Thesetherapeutic applications are described further in the followingsubsections.

Antibody Therapy

The cell surface nature and expression profiles of the STEAP's incancers including prostate cancer indicate that they are promisingtargets for antibody therapy of prostate and other cancers expressingSTEAP's. The experimental results described in the Examples hereinprovide compelling evidence that STEAP-1 is strongly expressed uniformlyover the surface of glandular epithelial cells within prostate andprostate cancer cells. In particular, immunohistochemical analysisresults show that the surface of human prostate epithelial cells (normaland cancer) appear to be uniformly coated with STEAP-1. Biochemicalanalysis confirms the cell surface localization of STEAP-1 initiallysuggested by its putative 6-transmembrane primary structural elementsand by the pericellular staining plainly visualized byimmunohistochemical staining.

STEAP-1 is uniformly expressed at high levels over the surface ofprostate glandular epithelia, an ideal situation for immunotherapeuticintervention strategies which target extracellular STEAP epitopes.Systemic administration of STEAP-immunoreactive compositions would beexpected to result in extensive contact of the composition with prostateepithelial cells via binding to STEAP-1 extracellular epitopes.Moreover, given the near absence of STEAP-1 protein expression in normalhuman tissues, there is ample reason to expect exquisite sensitivitywithout toxic, non-specific and/or non-target effects caused by thebinding of the immunotherapeutic composition to STEAP-1 on non-targetorgans and tissues.

In addition to the high level expression of STEAP-1 in prostate andprostate cancer cells, STEAP-1 appears to be substantiallyover-expressed in a variety of other human cancers, including bladder,colon, pancreatic and ovarian cancers. In particular, high level STEAP-1mRNA expression is detected in all tested prostate cancer tissues andcell lines, and in most of the pancreatic, colon, and bladder cancercell lines tested. High level expression of STEAP-1 is also observed insome ovarian cancer cell lines. Lower level expression is observed insome breast, testicular, and cervical cancer cell lines. Very high levelexpression is also detected in a Ewing sarcoma cell line. Applicantshave shown that cell surface STEAP-1 protein is expressed in bladder andcolon cancers, while there is no detectable cell surface (orintracellular) STEAP-1 protein in normal colon and low expression innormal bladder. Antibodies specifically reactive with extracellulardomains of STEAP-1 may be useful to treat these cancers systemically,either as toxin or therapeutic agent conjugates or as naked antibodiescapable of inhibiting cell proliferation or function.

STEAP-2 protein is also expressed in prostate cancer, and may beexpressed in other cancers as well. STEAP-2 mRNA analysis by RT-PCR andNorthern blot show that expression is restricted to prostate in normaltissues, is also expressed in some prostate, pancreatic, colon,testicular, ovarian and other cancers. Therefore, antibodies reactivewith STEAP-2 may be useful in the treatment of prostate and othercancers. Similarly, although not yet characterized by applicants, theexpression of STEAP-3 and STEAP-4 (as well as other STEAP's) may beassociated with some cancers. Thus antibodies reactive with these STEAPfamily member proteins may also be useful therapeutically.

STEAP antibodies may be introduced into a patient such that the antibodybinds to STEAP on the cancer cells and mediates the destruction of thecells and the tumor and/or inhibits the growth of the cells or thetumor. Mechanisms by which such antibodies exert a therapeutic effectmay include complement-mediated cytolysis, antibody-dependent cellularcytotoxicity, modulating the physiologic function of STEAP, inhibitingligand binding or signal transduction pathways, modulating tumor celldifferentiation, altering tumor angiogenesis factor profiles, and/or byinducing apoptosis. STEAP antibodies conjugated to toxic or therapeuticagents may also be used therapeutically to deliver the toxic ortherapeutic agent directly to STEAP-bearing tumor cells.

Cancer therapy using anti-STEAP antibodies may follow the teachingsgenerated from various approaches which have been successfully employedwith respect to other types of cancer, including but not limited tocolon cancer (Arlen, et al., Crit Rev Immunol (1998) 18:133-138),multiple myeloma (Ozaki, S., et al., Blood (1997) 90:3179-3186;Tsunenari, T., et al., Blood (1997) 90:2437-2444), gastric cancer(Kasprzyk, P. G., et al., Cancer Res. (1992) 52:2771-2776), B-celllymphoma (Funakoshi, S., et al., J. Immunother. (1996) 19:93-101),leukemia (Zhong, R., et al., Leuk. Res. (1996) 20:581-589), colorectalcancer (Mount, P. F., et al., Cancer Res. (1994) 54:6160-6166); Velders,M. P., et al., Cancer Res. (1995) 55:4398-4403), and breast cancer(Shepard, H. M., et al., J. Clin. Immunol. (1991) 11:117-127).

Although STEAP antibody therapy may be useful for all stages of theforegoing cancers, antibody therapy may be particularly appropriate andin advanced or metastatic cancers. Combining the antibody therapy methodof the invention with a chemotherapeutic or radiation regimen may bepreferred in patients who have not received chemotherapeutic treatment,whereas treatment with the antibody therapy of the invention may beindicated for patients who have received one or more chemotherapy.Additionally, antibody therapy may also enable the use of reduceddosages of concomitant chemotherapy, particularly in patients that donot tolerate the toxicity of the chemotherapeutic agent very well.

It may be desirable for non-prostate cancer patients to be evaluated forthe presence and level of STEAP over-expression, preferably usingimmunohistochemical assessments of tumor tissue, quantitative STEAPimaging, or other techniques capable of reliably indicating the presenceand degree of STEAP overexpression. Immunohistochemical analysis oftumor biopsies or surgical specimens may be preferred for this purpose.Methods for immunohistochemical analysis of tumor tissues are well knownin the art.

Anti-STEAP monoclonal antibodies useful in treating prostate and othercancers include those which are capable of initiating a potent immuneresponse against the tumor and those which are capable of directcytotoxicity. In this regard, anti-STEAP mAb's may elicit tumor celllysis by either complement-mediated or antibody-dependent cellcytotoxicity (ADCC) mechanisms, both of which require an intact Fcportion of the immunoglobulin molecule for interaction with effectorcell Fc receptor sites or complement proteins. In addition, anti-STEAPmAb's which exert a direct biological effect on tumor growth are usefulin the practice of the invention. Potential mechanisms by which suchdirectly cytotoxic mAb's may act include inhibition of cell growth,modulation of cellular differentiation, modulation of tumor angiogenesisfactor profiles, and the induction of apoptosis. The mechanism by whicha particular anti-STEAP mAb exerts an anti-tumor effect may be evaluatedusing any number of in vitro assays designed to determine ADCC, ADMMC,complement-mediated cell lysis, and so forth, as is generally known inthe art.

The anti-tumor activity of a particular anti-STEAP mAb, or combinationof anti-STEAP mAb's, may be evaluated in vivo using a suitable animalmodel. For example, xenogenic prostate cancer models wherein humanprostate cancer explants or passaged xenograft tissues are introducedinto immune compromised animals, such as nude or SCID mice, areappropriate in relation to prostate cancer and have been described(Klein, et al., Nature Medicine (1997) 3:402-408). For Example, PCTPatent Application WO 98/16628, Sawyers, et al., published Apr. 23,1998, describes various xenograft models of human prostate cancercapable of recapitulating the development of primary tumors,micrometastasis, and the formation of osteoblastic metastasescharacteristic of late stage disease. Efficacy may be predicted usingassays which measure inhibition of tumor formation, tumor regression ormetastasis, and the like.

It should be noted that the use of murine or other non-human monoclonalantibodies, human/mouse chimeric mAb's may induce moderate to strongimmune responses in some patients. In the most severe cases, such animmune response may lead to the extensive formation of immune complexeswhich, potentially, can cause renal failure. Accordingly, preferredmonoclonal antibodies used in the practice of the therapeutic methods ofthe invention are those which are either fully human or humanized andwhich bind specifically to the target 20P1F12/TMPRSS2 antigen with highaffinity but exhibit low or no antigenicity in the patient.

The method of the invention contemplates the administration of singleanti-STEAP mAb's as well as combinations, or “cocktails,” of differentmAb's. Such mAb cocktails may have certain advantages inasmuch as theycontain mAb's which exploit different effector mechanisms or combinedirectly cytotoxic mAb's with mAb's that rely on immune effectorfunctionality. Such mAb's in combination may exhibit synergistictherapeutic effects. In addition, the administration of anti-STEAP mAb'smay be combined with other therapeutic agents, including but not limitedto various chemotherapeutic agents, androgen-blockers, and immunemodulators (e.g., IL-2, GM-CSF). The anti-STEAP mAb's may beadministered in their “naked” or unconjugated form, or may havetherapeutic agents conjugated to them.

The anti-STEAP monoclonal antibodies used in the practice of the methodof the invention may be formulated into pharmaceutical compositionscomprising a carrier suitable for the desired delivery method. Suitablecarriers include any material which when combined with the anti-STEAPmAb's retains the anti-tumor function of the antibody and isnon-reactive with the subject's immune systems. Examples include, butare not limited to, any of a number of standard pharmaceutical carrierssuch as sterile phosphate buffered saline solutions, bacteriostaticwater, and the like.

The anti-STEAP antibody formulations may be administered via any routecapable of delivering the antibodies to the tumor site. Potentiallyeffective routes of administration include, but are not limited to,intravenous, intraperitoneal, intramuscular, intratumoral, intradermal,and the like. The preferred route of administration is by intravenousinjection. A preferred formulation for intravenous injection comprisesthe anti-STEAP mAb's in a solution of preserved bacteriostatic water,sterile unpreserved water, and/or diluted in polyvinylchloride orpolyethylene bags containing 0.9% sterile Sodium Chloride for Injection,USP. The anti-STEAP mAb preparation may be lyophilized and stored as asterile powder, preferably under vacuum, and then reconstituted inbacteriostatic water containing, for example, benzyl alcoholpreservative, or in sterile water prior to injection.

Treatment will generally involve the repeated administration of theanti-STEAP antibody preparation via an acceptable route ofadministration such as intravenous injection (IV), typically at a dosein the range of about 0.1 to about 10 mg/kg body weight. Doses in therange of 10-500 mg mAb per week may be effective and well tolerated.Based on clinical experience with the Herceptin® mAb in the treatment ofmetastatic breast cancer, an initial loading dose of approximately 4mg/kg patient body weight IV followed by weekly doses of about 2 mg/kgIV of the anti-STEAP mAb preparation may represent an acceptable dosingregimen. Preferably, the initial loading dose is administered as a 90minute or longer infusion. The periodic maintenance dose may beadministered as a 30 minute or longer infusion, provided the initialdose was well tolerated. However, as one of skill in the art willunderstand, various factors will influence the ideal dose regimen in aparticular case. Such factors may include, for example, the bindingaffinity and half life of the mAb or mAb's used, the degree of STEAPoverexpression in the patient, the extent of circulating shed STEAPantigen, the desired steady-state antibody concentration level,frequency of treatment, and the influence of chemotherapeutic agentsused in combination with the treatment method of the invention.

Optimally, patients should be evaluated for the level of circulatingshed STEAP antigen in serum in order to assist in the determination ofthe most effective dosing regimen and related factors. Such evaluationsmay also be used for monitoring purposes throughout therapy, and may beuseful to gauge therapeutic success in combination with evaluating otherparameters (such as serum PSA levels in prostate cancer therapy).

Cancer Vaccines

The invention further provides prostate cancer vaccines comprising aSTEAP protein or fragment thereof. The use of a tumor antigen in avaccine for generating humoral and cell-mediated immunity for use inanti-cancer therapy is well known in the art and has been employed inprostate cancer using human PSMA and rodent PAP immunogens (Hodge, etal., Int. J. Cancer (1995) 63:231-237; Fong, et al., J. Immunol. (1997)159:3113-3117). Such methods can be readily practiced by employing aSTEAP protein, or fragment thereof, or a STEAP-encoding nucleic acidmolecule and recombinant vectors capable of expressing and appropriatelypresenting the STEAP immunogen.

For example, viral gene delivery systems may be used to deliver aSTEAP-encoding nucleic acid molecule. Various viral gene deliverysystems which can be used in the practice of this aspect of theinvention include, but are not limited to, vaccinia, fowlpox, canarypox,adenovirus, influenza, poliovirus, adeno-associated virus, lentivirus,and Sindbis virus (Restifo, N. P., Curr. Opin. Immunol. (1996)8:658-663). Non-viral delivery systems may also be employed by usingnaked DNA encoding a STEAP protein or fragment thereof introduced intothe patient (e.g., intramuscularly) to induce an anti-tumor response. Inone embodiment, the full-length human STEAP cDNA may be employed. Inanother embodiment, STEAP nucleic acid molecules encoding specificcytotoxic T lymphocyte (CTL) epitopes may be employed. CTL epitopes canbe determined using specific algorithms (e.g., Epimer, Brown University)to identify peptides within a STEAP protein which are capable ofoptimally binding to specified HLA alleles. Optimally immunogenic HLAClass I molecule-binding peptides within the STEAP-1 and STEAP-2sequences have been analyzed in Example 9, below.

Various ex vivo strategies may also be employed. One approach involvesthe use of dendritic cells to present STEAP antigen to a patient'simmune system. Dendritic cells express MHC Class I and 11, B7costimulator, and IL-12, and are thus highly specialized antigenpresenting cells. In prostate cancer, autologous dendritic cells pulsedwith peptides of the prostate-specific membrane antigen (PSMA) are beingused in a Phase I clinical trial to stimulate prostate cancer patients'immune systems (Tjoa, B., et al., Prostate (1996) 28:65-69; Murphy, G.,et al., Prostate (1996) 29:371-380). Dendritic cells can be used topresent STEAP peptides to T cells in the context of MHC class I and IImolecules. In one embodiment, autologous dendritic cells are pulsed withSTEAP peptides capable of binding to MHC molecules. In anotherembodiment, dendritic cells are pulsed with the complete STEAP protein.Yet another embodiment involves engineering the overexpression of theSTEAP gene in dendritic cells using various implementing vectors knownin the art, such as adenovirus (Arthur, et al., Cancer Gene Ther. (1997)4:17-25), retrovirus (Henderson, et al., Cancer Res. (1996)56:3763-3770), lentivirus, adeno-associated virus, DNA transfection(Ribas, A., et al., Cancer Res. (1997) 57:2865-2869), and tumor-derivedRNA transfection (Ashley, D. M., et al., J. Exp. Med. (1997)186:1177-1182).

Anti-idiotypic anti-STEAP antibodies can also be used in anti-cancertherapy as a vaccine for inducing an immune response to cells expressinga STEAP protein. Specifically, the generation of anti-idiotypicantibodies is well known in the art and can readily be adapted togenerate anti-idiotypic anti-STEAP antibodies that mimic an epitope on aSTEAP protein (see, for example, Wagner, U., et al., Hybridoma (1997)16:33-40; Foon, K. A., et al., J. Clin. Invest. (1995) 96:334-342;Herlyn, D., et al., Cancer Immunol. Immunother. (1996) 43:65-76). Suchan anti-idiotypic antibody can be used in anti-idiotypic therapy aspresently practiced with other anti-idiotypic antibodies directedagainst tumor antigens.

Genetic immunization methods may be employed to generate prophylactic ortherapeutic humoral and cellular immune responses directed againstcancer cells expressing STEAP. Constructs comprising DNA encoding aSTEAP protein/immunogen and appropriate regulatory sequences may beinjected directly into muscle or skin of an individual, such that thecells of the muscle or skin take-up the construct and express theencoded STEAP protein/immunogen. Expression of the STEAP proteinimmunogen results in the generation of prophylactic or therapeutichumoral and cellular immunity against prostate cancer. Variousprophylactic and therapeutic genetic immunization techniques known inthe art may be used (for review, see information and referencespublished at Internet address www.genweb.com).

Kits

For use in the diagnostic and therapeutic applications described orsuggested above, kits are also provided by the invention. Such kits maycomprise a carrier means being compartmentalized to receive in closeconfinement one or more container means such as vials, tubes, and thelike, each of the container means comprising one of the separateelements to be used in the method. For example, one of the containermeans may comprise a probe which is or can be detectably labeled. Suchprobe may be an antibody or polynucleotide specific for a STEAP proteinor a STEAP gene or message, respectively. Where the kit utilizes nucleicacid hybridization to detect the target nucleic acid, the kit may alsohave containers containing nucleotide(s) for amplification of the targetnucleic acid sequence and/or a container comprising a reporter-means,such as a biotin-binding protein, such as avidin or streptavidin, boundto a reporter molecule, such as an enzymatic, florescent, orradionucleotide label.

EXAMPLES

Various aspects of the invention are further described and illustratedby way of the several examples which follow, none of which are intendedto limit the scope of the invention.

Example 1 Isolation of cDNA Fragment of STEAP-1 Gene Materials andMethods Cell Lines and Human Tissues

All human cancer cell lines used in this study were obtained from theATCC. All cell lines were maintained in DMEM with 10% fetal calf serum.PrEC (primary prostate epithelial cells) were obtained from Cloneticsand were grown in PrEBM media supplemented with growth factors(Clonetics).

All human prostate cancer xenografts were originally provided by CharlesSawyers (UCLA) (Klein, et al., 1997). LAPC-4 AD and LAPC-9 AD xenograftswere routinely passaged as small tissue chunks in recipient SCID males.LAPC-4 AI and LAPC-9 AI xenografts were derived as described previously(Klein, et al., 1997) and were passaged in castrated males or in femaleSCID mice. A benign prostatic hyperplasia tissue sample waspatient-derived.

Human tissues for RNA and protein analyses were obtained from the HumanTissue Resource Center (HTRC) at the UCLA (Los Angeles, Calif.) and fromQualTek, Inc. (Santa Barbara, Calif.).

RNA Isolation:

Tumor tissue and cell lines were homogenized in TRIzol® reagent (LifeTechnologies, Gibco-BRL) using 10 ml/g tissue or 10 ml/10⁸ cells toisolate total RNA. Poly A RNA was purified from total RNA using Qiagen'sOligotex mRNA Mini and Midi kits. Total and mRNA were quantified byspectrophotometric analysis (O.D. 260/280 nm) and analyzed by gelelectrophoresis.

Oligonucleotides:

The following HPLC purified oligonucleotides were used.

RSACDN (cDNA synthesis primer): 5′TTTTGTACAAGCTT₃₀3′ (SEQ ID NO: 22)Adaptor 1: 5′CTAATACGACTCACTATAGGGCTCGAGCGGCCGCCCGGGCAGGT3′ (SEQ ID NO:23) 3′GGCCCGTCCA5′ (SEQ ID NO: 24) Adaptor 2:5′GTAATACGACTCACTATAGGGCAGCGTGGTCGCGGCCGAGGT3′ (SEQ ID NO: 25)3′CGGCTCCA5′ (SEQ ID NO: 26) PCR primer 1: 5′CTAATACGACTCACTATAGGGC3′(SEQ ID NO: 27) Nested primer (NP)1: 5′TCGAGCGGCCGCCCGGGCAGGT3′ (SEQ IDNO: 28) Nested primer (NP)2: 5′AGCGTGGTCGCGGCCGAGGT3′ (SEQ ID NO: 29)Suppression Subtractive Hybridization:

Suppression Subtractive Hybridization (SSH) was used to identify cDNA'scorresponding to genes which may be up-regulated in androgen dependentprostate cancer compared to benign prostatic hyperplasia.

Double stranded cDNA's corresponding to the LAPC-4 AD xenograft (tester)and the BPH tissue (driver) were synthesized from 2 μg of poly(A)⁺ RNAisolated from xenograft and BPH tissue, as described above, usingClontech's PCR-Select cDNA Subtraction Kit and 1 ng of oligonucleotideRSACDN as primer. First- and second-strand synthesis were carried out asdescribed in the Kit's user manual protocol (Clontech Protocol No.PT1117-1, Catalog No. K1804-1). The resulting cDNA was digested with RsaI for 3 hrs. at 37° C. Digested cDNA was extracted withphenol/chloroform (1:1) and ethanol precipitated.

Driver cDNA (BPH) was generated by combining in a 4 to 1 ratio Rsa Idigested BPH cDNA with digested cDNA from mouse liver, in order toensure that murine genes were subtracted from the tester cDNA (LAPC-4AD).

Tester cDNA (LAPC-4 AD) was generated by diluting 1 μl of Rsa I digestedLAPC-4 AD cDNA (400 ng) in 5 μl of water. The diluted cDNA (2 μl, 160ng) was then ligated to 2 μl of adaptor 1 and adaptor 2 (10 μM), inseparate ligation reactions, in a total volume of 10 μl at 16° C.overnight, using 400 u of T4 DNA ligase (Clontech). Ligation wasterminated with 1 μl of 0.2 M EDTA and heating at 72° C. for 5 min.

The first hybridization was performed by adding 1.5 μl (600 ng) ofdriver cDNA to each of two tubes containing 1.5 μl (20 ng) adaptor 1-and adaptor 2-ligated tester cDNA. In a final volume of 4 μl, thesamples were overlaid with mineral oil, denatured in an MJ Research®thermal cycler at 98° C. for 1.5 minutes, and then were allowed tohybridize for 8 hrs at 68° C. The two hybridizations were then mixedtogether with an additional 1 μl of fresh denatured driver cDNA and wereallowed to hybridize overnight at 68° C. The second hybridization wasthen diluted in 200 μl of 20 mM Hepes, pH 8.3, 50 mM NaCl, 0.2 mM EDTA,heated at 70° C. for 7 min. and stored at −20° C.

PCR Amplification, Cloning and Sequencing of Gene Fragments Generatedfrom SSH:

To amplify gene fragments resulting from SSH reactions, two PCRamplifications were performed. In the primary PCR reaction 1 μl of thediluted final hybridization mix was added to 1 μl of PCR primer 1 (10μM), 0.5 μl dNTP mix (10 μM), 2.5 μl 10× reaction buffer (Clontech) and0.5 μl 50× Advantage cDNA polymerase Mix (Clontech) in a final volume of25 μl. PCR 1 was conducted using the following conditions: 75° C. for 5min., 94° C. for 25 sec., then 27 cycles of 94° C. for 10 sec, 66° C.for 30 sec, 72° C. for 1.5 min. Five separate primary PCR reactions wereperformed for each experiment. The products were pooled and diluted 1:10with water. For the secondary PCR reaction, 1 μl from the pooled anddiluted primary PCR reaction was added to the same reaction mix as usedfor PCR 1, except that primers NP1 and NP2 (10 μM) were used instead ofPCR primer 1. PCR 2 was performed using 10-12 cycles of 94° C. for 10sec, 68° C. for 30 sec, 72° C. for 1.5 minutes. The PCR products wereanalyzed using 2% agarose gel electrophoresis.

The PCR products were inserted into pCR2.1 using the T/A vector cloningkit (Invitrogen). Transformed E. coli were subjected to blue/white andampicillin selection. White colonies were picked and arrayed into 96well plates and were grown in liquid culture overnight. To identifyinserts, PCR amplification was performed on 1 ml of bacterial cultureusing the conditions of PCR 1 and NP1 and NP2 as primers. PCR productswere analyzed using 2% agarose gel electrophoresis.

Bacterial clones were stored in 20% glycerol in a 96 well format.Plasmid DNA was prepared, sequenced, and subjected to nucleic acidhomology searches of the GenBank, dbEST, and NCI-CGAP databases.

RT-PCR Expression Analysis:

First strand cDNA's were generated from 1 μg of mRNA with oligo(dT)12-18 priming using the Gibco-BRL Superscript Preamplificationsystem. The manufacturers protocol was used and included an incubationfor 50 min at 42° C. with reverse transcriptase followed by RNase Htreatment at 37° C. for 20 min. After completing the reaction, thevolume was increased to 200 μl with water prior to normalization. Firststrand cDNA's from 16 different normal human tissues were obtained fromClontech.

Normalization of the first strand cDNAs from multiple tissues wasperformed by using the primers 5′atatcgccgcgctcgtcgtcgacaa3′ (SEQ IDNO:36) and 5′agccacacgcagctcattgtagaagg 3′ (SEQ ID NO:37) amplifyβ-actin. First strand cDNA (5 μl) was amplified in a total volume of 50μl containing 0.4 μM primers, 0.2 μM each dNTPs, 1×PCR buffer (Clontech,10 mM Tris-HCL, 1.5 mM MgCl₂, 50 mM KCl, pH 8.3) and 1× KlenTaq DNApolymerase (Clontech). Five μl of the PCR reaction was removed at 18,20, and 22 cycles and used for agarose gel electrophoresis. PCR wasperformed using an MJ Research® thermal cycler under the followingconditions: initial denaturation was at 94° C. for 15 sec, followed by a18, 20, and 22 cycles of 94° C. for 15, 65° C. for 2 min, 72° C. for 5sec. A final extension at 72° C. was carried out for 2 min. Afteragarose gel electrophoresis, the band intensities of the 283 bp β-actinbands from multiple tissues were compared by visual inspection. Dilutionfactors for the first strand cDNAs were calculated to result in equalβ-actin band intensities in all tissues after 22 cycles of PCR. Threerounds of normalization were required to achieve equal band intensitiesin all tissues after 22 cycles of PCR.

To determine expression levels of the 8P1D4 gene, 5 μl of normalizedfirst strand cDNA was analyzed by PCR using 25, 30, and 35 cycles ofamplification using the following primer pairs:

5′ ACT TTG TTG ATG ACC AGG ATT GGA 3′ (SEQ ID NO: 14) 5′ CAG AAC TTC AGCACA CAC AGG AAC 3′ (SEQ ID NO: 15)products at cycle numbers that give light band intensities.Results:

Several SSH experiments were conduced as described in the Materials andMethods, supra, and led to the isolation of numerous candidate genefragment clones. All candidate clones were sequenced and subjected tohomology analysis against all sequences in the major public gene and ESTdatabases in order to provide information on the identity of thecorresponding gene and to help guide the decision to analyze aparticular gene for differential expression. In general, gene fragmentswhich had no homology to any known sequence in any of the searcheddatabases, and thus considered to represent novel genes, as well as genefragments showing homology to previously sequenced expressed sequencetags (EST's), were subjected to differential expression analysis byRT-PCR and/or Northern analysis.

One of the cDNA clones, designated 8P1D4, was 436 bp in length andshowed homology to an EST sequence in the NCI-CGAP tumor gene database.The full length cDNA encoding the 8P1D4 gene was subsequently isolatedusing this cDNA and re-named STEAP-1. The 8P1D4 cDNA nucleotide sequencecorresponds to nucleotide residues 150 through 585 in the STEAP-1 cDNAsequence as shown in FIG. 1A. Another clone, designated 28P3E1, 561 bpin length showed homology to a number of EST sequences in the NCI-CGAPtumor gene database or in other databases. Part of the 28P3E1 sequence(356 bp) is identical to an EST derived from human fetal tissue. Afterthe full length STEAP-1 cDNA was obtained and sequenced, it becameapparent that this clone also corresponds to STEAP-1 (more specifically,to residues 622 through the 3′ end of the STEAP-1 nucleotide sequence asshown in FIG. 1A).

Differential expression analysis by RT-PCR using primers derived fromthe 8P1D4 cDNA clone showed that the 8P1D4 (STEAP-1) gene is expressedat approximately equal levels in normal prostate and the LAPC-4 andLAPC-9 xenografts (FIG. 2, panel A). Further RT-PCR expression analysisof first strand cDNA's from 16 normal tissues showed greatest levels of8P1D4 expression in prostate. Substantially lower level expression inseveral other normal tissues (i.e., colon, ovary, small intestine,spleen and testis) was detectable only at 30 cycles of amplification inbrain, pancreas, colon and small intestine (FIG. 2, panels B and C).

Example 2 Isolation of Full Length STEAP-1 Encoding cDNA

The 436 bp 8P1D4 gene fragment (Example 1) was used to isolateadditional cDNA's encoding the 8P1D4/STEAP-1 gene. Briefly, a normalhuman prostate cDNA library (Clontech) was screened with a labeled probegenerated from the 436 bp 8P1D4 cDNA. One of the positive clones, clone10, is 1195 bp in length and encodes a 339 amino acid protein havingnucleotide and encoded amino acid sequences bearing no significanthomology to any known human genes or proteins (homology to a rat KidneyInjury Protein recently described in International Application WO98/53071). The encoded protein contains at least 6 predictedtransmembrane motifs implying a cell surface orientation (see FIG. 1A,predicted transmembrane motifs underlined). These structural featuresled to the designation “STEAP”, for “Six Transmembrane EpithelialAntigen of the Prostate”. Subsequent identification of additional STEAPproteins led to the re-designation of the 8P1D4 gene product as“STEAP-1”. The STEAP-1 cDNA and encoded amino acid sequences are shownin FIG. 1A and correspond to SEQ ID NOS: 1 and 2, respectively. STEAP-1cDNA clone 10 has been deposited with the American Type CultureCollection (“ATCC”) (Manassas, Va.) as plasmid 8P1D4 clone 10.1 on Aug.26, 1998 as ATCC Accession No. 98849. The STEAP-1 cDNA clone can beexcised therefrom using EcoRI/XbaI double digest (EcoRI at the 5′ end,XbaI at the 3′ end).

Example 3 STEAP-1 Gene and Protein Expression Analysis

In order to begin to characterize the biological characteristics ofSTEAP-1, an extensive evaluation of STEAP-1 mRNA and STEAP-1 proteinexpression across a variety of human tissue specimens was undertaken.This evaluation included Northern blot, Western blot andimmunohistochemical analysis of STEAP-1 expression in a large number ofnormal human tissues, human prostate cancer xenografts and cell lines,and various other human cancer cell lines.

Example 3A Northern Blot Analysis of STEAP-1 mRNA Expression in NormalHuman Tissues

Initial analysis of STEAP-1 mRNA expression in normal human tissues wasconducted by Northern blotting two multiple tissue blots obtained fromClontech (Palo Alto, Calif.), comprising a total of 16 different normalhuman tissues, using labeled STEAP-1 clone 10 as a probe. RNA sampleswere quantitatively normalized with a β-actin probe. The results areshown in FIG. 3A. The highest expression level was detected in normalprostate, with an approximately 5-10 fold lower level of expressiondetected in colon and liver. These northern blots showed two transcriptsof approximately 1.4 kb and 4.0 kb, the former of which corresponds tothe full length STEAP-1 clone 10 cDNA, which encodes the entire STEAP-1open reading frame. The larger transcript was separately cloned as a3627 bp cDNA from a normal prostate library, the sequence of whichcontains a 2399 bp intron (FIG. 4).

This initial analysis was extended by using the STEAP-1 clone 10 probeto analyze an RNA dot blot matrix of 37 normal human tissues (Clontech,Palo Alto, Calif.; Human Master Blot™). The results are shown in FIG. 3Band show strong STEAP-1 expression only in prostate. Very low levelSTEAP-1 RNA expression was detected in liver, lung, trachea and fetalliver tissue, at perhaps a 5-fold lower level compared to prostate. Noexpression was detected in any of the remaining tissues. Based on theseanalyses, significant STEAP-1 expression appears to be prostate specificin normal tissues.

Example 3B Northern Blot Analysis of STEAP-1 mRNA Expression in ProstateCancer Xenografts and Cell Lines

To analyze STEAP-1 expression in human cancer tissues and cell lines,RNA's derived from human prostate cancer xenografts and an extensivepanel of prostate and non-prostate cancer cell lines were analyzed byNorthern blot using STEAP-1 cDNA clone 10 as probe. All RNA samples werequantitatively normalized by ethidium bromide staining and subsequentanalysis with a labeled β-actin probe.

The results, presented in FIG. 5, show high level STEAP-1 expression inall the LAPC xenografts and all of the prostate cancer cell lines.Expression in the LAPC-9 xenografts was higher compared to the LAPC-4xenografts, with no significant difference observed betweenandrogen-dependent and androgen-independent sublines (FIG. 5A).Expression in the LAPC-4 xenografts was comparable to expression innormal prostate. Lower levels of expression were detected in PrEC cells(Clonetics), which represent the basal cell compartment of the prostate.Analysis of prostate cancer cell lines showed highest expression levelsin LNCaP, an androgen dependent prostate carcinoma cell line.Significant expression was also detected in the androgen-independentcell lines PC-3 and DU145. High levels of STEAP expression were alsodetected in LAPC-4 and LAPC-9 tumors that were grown within the tibia ofmice as a model of prostate cancer bone metastasis (FIG. 5B).

Significantly, very strong STEAP-1 expression was also detected in manyof the non-prostate human cancer cell lines analyzed (FIG. 5A).Particularly high level expression was observed in RD-ES cells, an Ewingsarcoma (EWS) derived cell line. Additionally, very high levelexpression was also detected in several of the colon cancer cell lines(e.g., CaCo-2, LoVo, T84 and Colo-205), bladder carcinoma cell lines(e.g., SCABER, UM-UC-3, TCCSUP and 5637), ovarian cancer cell lines(e.g., OV-1063 and SW 626) and pancreatic cancer cell lines (e.g., HPAC,Capan-1, PANC-1 and BxPC-3). These results, combined with the absence ofstrong expression in the corresponding normal tissues (FIG. 3), indicatethat STEAP-1 may be generally up-regulated in these types (as well asother types) of human cancers.

Example 3C Western Blot Analysis of STEAP-1 Protein Expression inProstate and Other Cancers

A 15 mer peptide corresponding to amino acid residues 14 through 28 ofthe STEAP-1 amino acid sequence as shown in FIG. 1A (WKMKPRRNLEEDDYL)(SEQ ID NO: 35) was synthesized and used to immunize sheep for thegeneration of sheep polyclonal antibodies towards the amino-terminus ofthe protein (anti-STEAP-1) as follows. The peptide was conjugated to KLH(keyhole limpet hemocyanin). The sheep was initially immunized with 400μg of peptide in complete Freund's adjuvant. The animal was subsequentlyboosted every two weeks with 200 μg of peptide in incomplete Freund'sadjuvant. Anti-STEAP antibody was affinity-purified from sheep serumusing STEAP peptide coupled to Affi-Gel 10 (Bio-Rad). Purified antibodyis stored in phosphate-buffered saline with 0.1% sodium azide.

To test antibody specificity, the cDNA of STEAP-1 was cloned into aretroviral expression vector (pSRαtkneo, Muller, A. J., et al., Mol.Cell. Biol. (1991) 11:1785-1792). National Institutes of Health (NIH)3T3 cells were infected with retroviruses encoding STEAP-1 and wereselected in G418 for 2 weeks. Western blot analysis of protein extractsof infected and un-infected NIH 3T3 cells showed expression of a proteinwith an apparent molecular weight of 36 kD only in the infected cells(FIG. 6, lanes marked “3T3 STEAP” AND “3T3”).

The anti-STEAP-1 polyclonal antibody was used to probe Western blots ofcell lysates prepared from a variety of prostate cancer xenografttissues, prostate cancer cell lines and other non-prostate cancer celllines. Protein samples (20 μg each) were quantitatively normalized byprobing the blots with an anti-Grb-2 antibody.

The results are shown in FIG. 6. STEAP-1 protein was detected in all ofthe LAPC prostate cancer xenografts, all of the prostate cancer celllines, a primary prostate cancer specimen and its matched normalprostate control. Highest STEAP-1 protein expression was detected in theLAPC-9 xenograft and in LNCaP cells, in agreement with the Northern blotanalysis described immediately above. High level expression was alsoobserved in the bladder carcinoma cell line UM-UC-3. Expression in othercancer cell lines was also detectable (FIG. 6).

Example 3D Immunohistochemical Analysis of STEAP-1 Protein Expression inProstate Tumor Biopsy and Surgical Specimens

To determine the extent of STEAP-1 protein expression in clinicalmaterials, tissue sections were prepared from a variety of prostatecancer biopsies and surgical samples for immunohistochemical analysis.Tissues were fixed in 10% formalin, embedded in paraffin, and sectionedaccording to standard protocol. Formalin-fixed, paraffin-embeddedsections of LNCaP cells were used as a positive control. Sections werestained with an anti-STEAP-1 polyclonal antibody directed against aSTEAP-1 N-terminal epitope (as described immediately above). LNCaPsections were stained in the presence of an excess amount of the STEAP-1N-terminal peptide immunogen used to generate the polyclonal antibody(peptide 1) or a non-specific peptide derived from a distinct region ofthe STEAP-1 protein (peptide 2; YQQVQQNKEDAWIEH) (SEQ ID NO: 30).

The results are shown in FIG. 8. LNCaP cells showed uniformly strongperi-cellular staining in all cells (FIG. 8 b). Excess STEAP N-terminalpeptide (peptide 1) was able to competitively inhibit antibody staining(FIG. 8 a), while peptide 2 had no effect (FIG. 8 b). Similarly,uniformly strong peri-cellular staining was seen in the LAPC-9 (FIG. 8f) and LAPC-4 prostate cancer xenografts (data not shown). These resultsare clear and suggest that the staining is STEAP specific. Moreover,these results visually localize STEAP to the plasma membrane,corroborating the biochemical findings presented in Example 4 below.

The results obtained with the various clinical specimens are show inFIG. 8 c (normal prostate tissue), FIG. 8 d (grade 3 prostaticcarcinoma), and FIG. 8 e (grade 4 prostatic carcinoma), and are alsoincluded in the summarized results shown in Table 1. Light to strongstaining was observed in the glandular epithelia of all prostate cancersamples tested as well as in all samples derived from normal prostate orbenign disease. The signal appears to be strongest at the cell membraneof the epithelial cells, especially at the cell-cell junctions (FIGS. 8c, d and e) and is also inhibited with excess STEAP N-terminal peptide 1(data not shown). Some basal cell staining is also seen in normalprostate (FIG. 8 c), which is more apparent when examining atrophicglands (data not shown). STEAP-1 seems to be expressed at all stages ofprostate cancer since lower grades (FIG. 8 d), higher grades (FIG. 8 e)and metastatic prostate cancer (represented by LAPC-9; FIG. 8 f) allexhibit strong staining.

Immunohistochemical staining of a large panel of normal non-prostatetissues showed no detectable STEAP-1 expression in 24 of 27 of thesenormal tissues (Table 1). Only three tissue samples showed some degreeof anti-STEAP-1 staining. In particular, normal bladder exhibited lowlevels of cell surface staining in the transitional epithelium (FIG. 8g). Pancreas and pituitary showed low levels of cytoplasmic staining(Table 1). It is unclear whether the observed cytoplasmic staining isspecific or is due to non-specific binding of the antibody, sincenorthern blotting showed little to no STEAP-1 expression in pancreas(FIG. 3). Normal colon, which exhibited higher mRNA levels than pancreasby Northern blotting (FIG. 3), exhibited no detectable staining withanti-STEAP antibodies (FIG. 8 h). These results indicate that cellsurface expression of STEAP-1 in normal tissues appears to be restrictedto prostate and bladder.

TABLE 1 Immunohistochemical Staining of Human Tissues With Anti-STEAP-1Polyclonal Antibody STAINING INTENSITY TISSUE NONE cerebellum, cerebralcortex, spinal cord, heart, skeletal muscle, artery, thymus, spleen,bone marrow, lymph node, lung, colon, liver, stomach, kidney, testis,ovary, fallopian tubes, placenta, uterus, breast, adrenal gland, thyroidgland, skin, bladder (3/5) LIGHT TO bladder (2/5), pituitary gland(cytoplasmic), pancreas MODERATE (cytoplasmic), BPH (3/5), prostatecancer (3/10) STRONG prostate (2/2), BPH (2/5), prostate cancer** (7/10)*In cases where more than one sample is analyzed per tissue, the numbersin brackets indicates how many samples correspond to the stainingcategory/total analyzed. **Prostate cancer grades ranged from Gleasongrades 3 to 5.

Example 4 Biochemical Characterization of STEAP-1 Protein

To initially characterize the STEAP-1 protein, cDNA clone 10 was clonedinto the pcDNA 3.1 Myc-His plasmid (Invitrogen), which encodes a 6-Histag at the carboxyl-terminus, transfected into 293T cells, and analyzedby flow cytometry using anti-His monoclonal antibody (His-probe, SantaCruz) as well as the anti-STEAP-1 polyclonal antibody described above.Staining of cells was performed on intact cells as well as permeabilizedcells. The results indicated that only permeabilized cells stained withboth antibodies, suggesting that both termini of the STEAP-1 protein arelocalized intracellularly. It is therefore possible that one or more ofthe STEAP-1 protein termini are associated with intracellular organellesrather than the plasma membrane.

To determine whether STEAP-1 protein is expressed at the cell surface,intact STEAP-1-transfected 293T cells were labeled with a biotinylationreagent that does not enter live cells. STEAP-1 was thenimmunoprecipitated from cell extracts using the anti-His and anti-STEAPantibodies. SV40 large T antigen, an intracellular protein that isexpressed at high levels in 293T cells, and the endogenous cell surfacetransferrin receptor were immunoprecipitated as negative and positivecontrols, respectively. After immunoprecipitation, the proteins weretransferred to a membrane and visualized with horseradishperoxidase-conjugated streptavidin. The results of this analysis areshown in FIG. 7. Only the transferrin receptor (positive control) andSTEAP-1 were labeled with biotin, while the SV40 large T antigen(negative control) was not detectably labeled (FIG. 7A). Since only cellsurface proteins are labeled with this technique, it is clear from theseresults that STEAP-1 is a cell surface protein. Combined with theresults obtained from the flow cytometric analysis, it is clear thatSTEAP-1 is a cell surface protein with intracellular amino- andcarboxyl-termini.

Furthermore, the above results together with the STEAP-1 secondarystructural predictions, shows that STEAP-1 is a type IIIa membraneprotein with a molecular topology of six potential transmembranedomains, three extracellular loops, two intracellular loops and twointracellular termini. A schematic representation of STEAP-1 proteintopology relative to the cell membrane is shown in FIG. 1B.

In addition, prostate, bladder and colon cancer cells were directlyanalyzed for cell surface expression of STEAP-1 by biotinylationstudies. Briefly, biotinylated cell surface proteins were affinitypurified with streptavidin-gel and probed with the anti-STEAP-1polyclonal antibody described above. Western blotting of thestreptavidin purified proteins clearly show cell surface biotinylationof endogenous STEAP-1 in all prostate (LNCaP, PC-3, DU145), bladder(UM-UC-3, TCCSUP) and colon cancer (LoVo, Colo) cells tested, as well asin NIH 3T3 cells infected with a STEAP-1 encoding retrovirus, but not innon-expressing NIH 3T3 cells used as a negative control (FIG. 7B). In afurther negative control, STEAP-1 protein was not detected instreptavidin precipitates from non-biotinylated, STEAP expressing cells(FIG. 7B).

Example 5 Identification and Structural Analysis Other Human STEAP's

STEAP-1 has no homology to any known human genes. In an attempt toidentify additional genes that are homologous to STEAP-1, the proteinsequence of STEAP-1 was used as an electronic probe to identify familymembers in the public EST (expression sequence tag) database (dbEST).Using the “tblastn” function in NCBI (National Center for BiotechnologyInformation), the dbEST database was queried with the STEAP-1 proteinsequence. This analysis revealed additional putative STEAP-1 homologuesor STEAP family members, as further described below.

In addition, SSH cloning experiments also identified a STEAP-1 relatedcDNA fragment, clone 98P4B6. This clone was isolated from SSH cloningusing normal prostate cDNA as tester and LAPC-4 AD cDNA as driver. Alarger partial sequence of the 98P4B6 clone was subsequently isolatedfrom a normal prostate library; this clone encodes an ORF of 173 aminoacids with close homology to the primary structure of STEAP-1, and thuswas designated STEAP-2. A full length STEAP-2 cDNA of 2454 bp wasisolated from a prostate library. The STEAP-2 nucleotide and encoded ORFamino acid sequences are shown in FIG. 9. An amino acid alignment of theSTEAP-1 and partial STEAP-2 primary structures is shown in FIGS. 11A and11B. STEAP-1 and -2 share 61% identity over their 171 amino acid residueoverlap (FIG. 11B). The STEAP-2 cDNA has been deposited with theAmerican Type Culture Collection (“ATCC”) (Manassas, Va.) as plasmid98P4B6-GTD3 as ATCC Accession No. PTA-311.

These deposits were made under the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure and the Regulations thereunder (BudapestTreaty). This assures maintenance of a viable culture of the deposit for30 years from the date of deposit and for at least five (5) years afterthe most recent request for the furnishing of a sample of the depositreceived by the depository. The deposits will be made available by ATCCunder the terms of the Budapest Treaty, and subject to an agreementbetween Genentech, Inc. and ATCC, which assures that all restrictionsimposed by the depositor on the availability to the public of thedeposited material will be irrevocably removed upon the granting of thepertinent U.S. patent, assures permanent and unrestricted availabilityof the progeny of the culture of the deposit to the public upon issuanceof the pertinent U.S. patent or upon laying open to the public of anyU.S. or foreign patent application, whichever comes first, and assuresavailability of the progeny to one determined by the U.S. Commissionerof Patents and Trademarks to be entitled thereto according to 35 USC §122 and the Commissioner's rules pursuant thereto (including 37 CFR §1.14 with particular reference to 886 OG 638).

The STEAP-2 cDNA (98P4B6-GTD3) contains a 355 bp 5′UTR (untranslatedregion) that is 72% GC rich, suggesting that it contains translationalregulatory elements. The cDNA encodes an open reading frame (ORF) of 454amino acids (a.a.) with six potential transmembrane domains. This is incontrast to STEAP-1, which is 339 a.a. in length. Alignment with STEAP-1demonstrates 54.9% identity over a 237 amino acid overlap.Interestingly, the locations of the six putative transmembrane domainsin STEAP-2 coincide with the locations of the transmembrane domains inSTEAP-1 (see alignment). The homology of STEAP-2 with STEAP-1 is highestin the regions spanned by the first putative extracellular loop to thefifth transmembrane domain. This analysis and the sequence of STEAP-2suggest some significant differences between STEAP-1 and STEAP-2:STEAP-2 exhibits a 205 a.a. long intracellular N-terminus (compared to69 a.a. in STEAP-1) and a short 4 a.a. intracellular C-terminus(compared to 26 a.a. in STEAP-1). These differences could implysignificant differences in function and/or interaction withintracellular signaling pathways. To identify a unique mouse ESTcorresponding to STEAP-2, the unique N-terminus of STEAP-2 was used toquery the dbEST database. One mouse EST was isolated (AI747886, mousekidney) that may be used in the identification of mouse STEAP-2 and inexpression analysis of STEAP-2 in mouse.

Two EST's encoding ORF's bearing close homology to the STEAP-1 andSTEAP-2 sequences were also identified by electronic probing with theSTEAP-1 protein sequence. These EST's (AI139607 and R80991) wereprovisionally designated STEAP-3 and STEAP-4. A full length cDNAencoding STEAP-3 was subsequently cloned, and its nucleotide and deducedamino acid sequences are shown in FIG. 10A. The nucleotide sequences ofthe EST's corresponding to the STEAP's are reproduced in FIG. 10B.

An amino acid alignment of the structures of STEAP-1, STEAP-2, STEAP-3and the partial sequence of the putative STEAP-4 is shown in FIG. 11A.This alignment shows a close structural similarity between all fourSTEAP family proteins, particularly in the predicted transmembranedomains. As indicated above, STEAP-1 and STEAP-2 demonstrate 54.9%identity over a 237 amino acid overlap. STEAP-1 and STEAP-3 are 40.9%identical over a 264 amino acid region, while STEAP-2 and STEAP-3 are47.8% identical over a 416 amino acid region.

Example 6 Expression Analysis of STEAP-2 and Other Human STEAP FamilyMembers Example 6A Tissue Specific Expression of STEAP Family Members inNormal Human Tissues

RT-PCR analysis of STEAP-2 shows expression in all the LAPC prostatecancer xenografts and in normal prostate (FIG. 14, panel A). Analysis of8 normal human tissues shows prostate-specific expression after 25cycles of amplification (FIG. 14, panel B). Lower level expression inother tissues was detected only after 30 cycles of amplification.Northern blotting for STEAP-2 shows a pattern of 2 transcripts(approximately 3 and 8 kb in size) expressed only in prostate (and atsignificantly lower levels in the LAPC xenografts), with no detectableexpression in any of the 15 other normal human tissues analyzed (FIG.15, panel C). Thus, STEAP-2 expression in normal human tissues appearsto be highly prostate-specific.

Expression analysis of STEAP family members in normal tissues wasperformed by Northern blot and/or RT-PCR. All STEAP family membersappeared to exhibit tissue restricted expression patterns.STEAP-3/AI139607 expression is shown in FIG. 12A (Northern) and FIG. 12B(RT-PCR). STEAP-4/R80991 expression is shown in FIG. 13.

Example 6B Expression of STEAP-2 in Various Cancer Cell Lines

The RT-PCR results above suggested that the different STEAP familymembers exhibit different tissue expression patterns. Interestingly,STEAP-2, which appears very prostate-specific, seems to be expressed atlower levels in the LAPC xenografts. This is in contrast to STEAP-1,which is highly expressed in both normal and malignant prostate tissue.

To better characterize this suggested difference in the STEAP-2 prostatecancer expression profile (relative to STEAP-1), Northern blotting wasperformed on RNA derived from the LAPC xenografts, as well as severalprostate and other cancer cell lines, using a STEAP-2 specific probe(labeled cDNA clone 98P4B6). The results are shown in FIG. 16 and can besummarized as follows. STEAP-2 is highly expressed in normal prostateand in some of the prostate cancer xenografts and cell lines. Moreparticularly, very strong expression was observed in the LAPC-9 ADxenograft and the LNCaP cells. Significantly attenuated or no expressionwas observed in the other prostate cancer xenografts and cell lines.Very strong expression was also evident in the Ewing sarcoma cell lineRD-ES. Unlike STEAP-1, which is highly expressed in cancer cell linesderived from bladder, colon, pancreatic and ovarian tumors, STEAP-2showed low to non-detectable expression in these same cell lines(compare with FIG. 5). Interestingly, STEAP-2 was also non-detectable inPrEC cells, which are representative of the normal basal cellcompartment of the prostate. These results suggest that expression ofSTEAP-1 and STEAP-2 are differentially regulated. While STEAP-1 may be agene that is generally up-regulated in cancer, STEAP-2 may be a genethat is more restricted to normal prostate and prostate cancer.

Example 7 Chromosomal Localization of STEAP Genes

The chromosomal localization of STEAP-1 was determined using theGeneBridge 4 Human/Hamster radiation hybrid (RH) panel (Walter, M. A.,et al., Nat. Genet. (1994) 7:22-28) (Research Genetics, HuntsvilleAla.), while STEAP-2 and the STEAP homologues were mapped using theStanford G3 radiation hybrid panel (Stewart, E., et al., Genome Res.(1997) 7:422-433).

The following PCR primers were used for STEAP-1:

8P1D4.1 5′ ACTTTGTTGATGACCAGGATTGGA 3′ (SEQ ID NO: 14) 8P1D4.2 5′CAGAACTTCAGCACACACAGGAAC 3′ (SEQ ID NO: 15)

The resulting STEAP-1 mapping vector for the 93 radiation hybrid panelDNAs(210000020110101000100000010111010122100011100111011010100010001000101001021000001111001010000), and the mapping program RHMapper available at theBroad Institute website, localized the STEAP-1 gene to chromosome7p22.3, telomeric to D7S531.

The following PCR primers were used for 98P4B6/STEAP-2:

98P4B6.1 5′ GACTGAGCTGGAACTGGAATTTGT 3′ (SEQ ID NO: 31) 98P4B6.2 5′TTTGAGGAGACTTCATCTCACTGG 3′ (SEQ ID NO: 32)

The resulting vector (00000100100000000000000000000000100100000000001000100000000000001000010101010010011), and the mapping program RHserveravailable at the website for the Stanford Human Genome Center maps the98P4B6 (STEAP-2) gene to chromosome 7q21.

The following PCR primers were used for AI139607:

AI139607.1 5′ TTAGGACAACTTGATCACCAGCA 3′ (SEQ ID NO: 16) AI139607.2 5′TGTCCAGTCCAAACTGGGTTATTT 3′ (SEQ ID NO: 17)

The resulting vector (00000000100000000000000000001000100000200000001000100000001000000100010001010000010), and the mapping program RHserveravailable at the website for the Stanford Human Genome Center mapsAI139607 to chromosome 7q21.

The following PCR primers were used for R80991:

R80991.3 5′ ACAAGAGCCACCTCTGGGTGAA 3′ (SEQ ID NO: 33) R80991.4 5′AGTTGAGCGAGTTTGCAATGGAC 3′ (SEQ ID NO: 34)

The resulting vector (00000000000200001020000000010000000000000000000010000000001000011100000001001000001), and the mapping program RHserveravailable at the website for the Stanford Human Genome Center mapsR80991 to chromosome 2q14-q21, near D2S2591.

In summary, the above results show that three of the putative humanSTEAP family members localize to chromosome 7, as is schematicallydepicted in FIG. 17. In particular, the STEAP-1 gene localizes to thefar telomeric region of the short arm of chromosome 7, at 7p22.3, whileSTEAP-2 and AI139607 localize to the long arm of chromosome 7, at 7q21(FIG. 17). R80991 maps to chromosome 2q14-q21.

Example 8 Identification of Intron-Exon Boundaries of STEAP-1

Genomic clones for STEAP-1 were identified by searching GenBank for BACclones containing STEAP-1 sequences, resulting in the identification ofaccession numbers AC004969 (PAC DJ1121E10) and AC005053 (BAC RG041D11).Using the sequences derived from the PAC and BAC clones for STEAP theintron-exon boundaries were defined (FIG. 18). A total of 4 exons and 3introns were identified within the coding region of the STEAP gene.Knowledge of the exact exon-intron structure of the STEAP-1 gene may beused for designing primers within intronic sequences which in turn maybe used for genomic amplification of exons. Such amplification permitssingle-stranded conformational polymorphism (SSCP) analysis to searchfor polymorphisms associated with cancer. Mutant or polymorphic exonsmay be sequenced and compared to wild type STEAP. Such analysis may beuseful to identify patients who are more susceptible to aggressiveprostate cancer, as well as other types of cancer, particularly colon,bladder, pancreatic, ovarian, cervical and testicular cancers.

Southern blot analysis shows that the STEAP-1 gene exists in severalspecies including mouse (FIG. 19). Therefore, a mouse BAC library (MouseES 129-V release I, Genome Systems, FRAC-4431) was screened with thehuman cDNA for STEAP-1 (clone 10, Example 2). One positive clone, 12P11,was identified and confirmed by southern blotting (FIG. 20). Theintron-exon boundary information for human STEAP may be used to identifythe mouse STEAP-1 coding sequences.

The mouse STEAP-1 genomic clone may be used to study the biological roleof STEAP-1 during development and tumorigenesis. Specifically, the mousegenomic STEAP-1 clone may be inserted into a gene knock-out (K/O) vectorfor targeted disruption of the gene in mice, using methods generallyknown in the art. In addition, the role of STEAP in metabolic processesand epithelial cell function may be elucidated. Such K/O mice may becrossed with other prostate cancer mouse models, such as the TRAMP model(Greenberg, N. M., et al., Proc. Natl. Acad. Sci. USA (1995) 92:3439),to determine whether STEAP influences the development and progression ofmore or less aggressive and metastatic prostate cancers.

Example 9 Prediction of HLA-A2 Binding Peptides from STEAP-1 and STEAP-2

The complete amino acid sequences of the STEAP-1 and STEAP-2 proteinswere entered into the HLA Peptide Motif Search algorithm found in theBioinformatics and Molecular Analysis Section (BIMAS) Web site. The HLAPeptide Motif Search algorithm was developed by Dr. Ken Parker based onbinding of specific peptide sequences in the groove of HLA Class Imolecules and specifically HLA-A2 (Falk et al., 1991, Nature 351: 290-6;Hunt et al., 1992, Science 255:1261-3; Parker et al., 1992, J. Immunol.149:3580-7; Parker et al., 1994, J. Immunol. 152:163-75). This algorithmallows location and ranking of 8-mer, 9-mer, and 10-mer peptides from acomplete protein sequence for predicted binding to HLA-A2 as well asother HLA Class I molecules. Most HLA-A2 binding peptides are 9-mersfavorably containing a leucine (L) at position 2 and a valine (V) orleucine (L) at position 9.

The results of STEAP-1 and STEAP-2 predicted binding peptides are shownin Table 2 below. For both proteins the top 5 ranking candidates areshown along with their location, the amino acid sequence of eachspecific peptide, and an estimated binding score. The binding scorecorresponds to the estimated half-time of dissociation of complexescontaining the peptide at 37° C. at pH 6.5. Peptides with the highestbinding score (i.e., 10776.470 for STEAP-1 peptide 165; 1789.612 forSTEAP-2 peptide 227) are predicted to be the most tightly bound to HLAClass I on the cell surface and thus represent the best immunogenictargets for T-cell recognition. Actual binding of peptides to HLA-A2 canbe evaluated by stabilization of HLA-A2 expression on theantigen-processing defective cell line T2 (Refs. 5,6). Immunogenicity ofspecific peptides can be evaluated in vitro by stimulation of CD8+cytotoxic T lymphocytes (CTL) in the presence of dendritic cells (Xue,B. H., et al., Prostate (1997) 30:73-78; Peshwa, M. V., et al., Prostate(1998) 36:129-138).

TABLE 2 Predicted Binding of STEAP-1 and STEAP-2 Peptide Sequences withHighest Affinity for HLA-A2 Score (Estimate of Half Time of StartDisassociation of a Molecule Rank Position Subsequence Residue Listingcontaining This Subsequence STEAP-1 1 165 GLLSFFFAV (SEQ ID NO: 38)10776.470 2  86 FLYTLLREV (SEQ ID NO: 39) 470.951 3 262 LLLGTIHAL (SEQID NO: 40) 309.050 4 302 LIFKSILFL (SEQ ID NO: 41) 233.719 5 158MLTRKQFGL (SEQ ID NO: 42) 210.633 STEAP-2 1 227 FLYSFVRDV (SEQ ID NO:43) 1789.612 2 402 ALLISTFHV (SEQ ID NO: 44) 1492.586 3 307 LLSFFFAMV(SEQ ID NO: 45) 853.681 4 306 GLLSFFFAM (SEQ ID NO: 46) 769.748 5 100SLWDLRHLL (SEQ ID NO: 47) 726.962

This application is a continuation-in-part of U.S. application Ser. No.09/323,873 filed Jun. 1, 1999, entitled “NOVEL SERPENTINE TRANSMEMBRANEANTIGENS EXPRESSED 1N HUMAN CANCERS AND USES THEREOF” The proteinsdesignated “STRAP” in that application have been re-named “STEAP” in thepresent application.

Throughout this application, various publications are referenced withinparentheses. The disclosures of these publications are herebyincorporated by reference herein in their entireties.

The present invention is not to be limited in scope by the embodimentsdisclosed herein, which are intended as single illustrations ofindividual aspects of the invention, and any which are functionallyequivalent are within the scope of the invention. Various modificationsto the models and methods of the invention, in addition to thosedescribed herein, will become apparent to those skilled in the art fromthe foregoing description and teachings, and are similarly intended tofall within the scope of the invention. Such modifications or otherembodiments can be practiced without departing from the true scope andspirit of the invention.

1. An assay for determining the presence of cancer in a subject thatexpresses SEQ ID NO:5 gene product, comprising: providing a test sampleobtained from a subject having or suspected of having a cancer cellexpressing the SEQ ID NO:5 gene product, and a normal sample;determining the expression level of the SEQ ID NO:5 gene product in thetest sample and the normal sample; and comparing the expression level ofthe SEQ ID NO:5 gene product detected in the test sample and the normalsample, wherein a significant increase in SEQ ID NO:5 gene productexpression in the test sample relative to expression levels in thecorresponding normal sample is indicative of cancer.
 2. The assay ofclaim 1, wherein the test sample and the normal sample are obtained fromthe same subject.
 3. The assay of claim 1, wherein the test sample isobtained from the subject and the normal sample is obtained from anormal subject.
 4. The assay of claim 1, wherein the test sample isobtained from a prostate.
 5. An immunoassay for determining the presenceof cancer in a subject that expresses SEQ ID NO:6 in a test sample,comprising: providing a test sample obtained from a subject having orsuspected of having a cancer cell expressing SEQ ID NO:6; contacting thetest sample and the normal sample with an antibody or fragment thereofthat binds specifically to SEQ ID NO:6 or an extracellular domain of SEQID NO:6; quantifying an amount of SEQ ID NO:6 bound to said antibody orfragment thereof in the test sample and the normal sample; and comparingthe amount of SEQ ID NO:6 bound to said antibody or fragment thereof inthe test sample and the normal sample, whereby expression levels of theSTEAP-2 detected in the test sample and the normal sample are assessed,wherein a significant increase in SEQ ID NO:6 expression level in thetest sample relative to the corresponding normal sample is indicative ofcancer.
 6. The immunoassay of claim 5, wherein the antibody or fragmentthereof is a monoclonal antibody.
 7. The immunoassay of claim 5, whereinthe antibody or fragment thereof is labeled with a detectable marker. 8.The immunoassay of claim 5, wherein the antibody or fragment thereof isan Fab, F(ab′)2, Fv or sFv fragment.
 9. The immunoassay of claim 5,wherein the antibody or fragment thereof is a human antibody, ahumanized antibody, or a chimeric antibody.
 10. The immunoassay of claim5, wherein the test sample is obtained from a prostate.
 11. Theimmunoassay of claim 5, wherein the test sample and the normal sampleare obtained from the same subject.
 12. The immunoassay of claim 5,wherein the test sample is obtained from the subject and the normalsample is obtained from a normal subject.
 13. The method of claim 5,wherein the antibody or fragment thereof is recombinantly produced.